High affinity human antibodies to human angiopoietin-2

ABSTRACT

The present invention provides antibodies that bind to angiopoietin-2 (Ang-2) and methods of using same. According to certain embodiments of the invention, the antibodies are fully human antibodies that bind to human Ang-2. The antibodies of the invention are useful, inter alia, for the treatment of diseases and disorders associated with one or more Ang-2 biological activities including angiogenesis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.provisional application No. 61/229,418, filed on Jul. 29, 2009; and61/295,194, filed on Jan. 15, 2010, the disclosures of which are hereinincorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to antibodies, and antigen-bindingfragments thereof, which are specific for angiopoietin-2 (Ang-2).

BACKGROUND

Angiogenesis is the biological process whereby new blood vessels areformed. Aberrant angiogenesis is associated with several diseaseconditions including, e.g., proliferative retinopathies, rheumatoidarthritis and psoriasis. In addition, it is well established thatangiogenesis is critical for tumor growth and maintenance.Angiopoietin-2 (Ang-2) is a ligand for the Tie-2 receptor (Tie-2) andhas been shown to play a role in angiogenesis. Ang-2 is also referred toin the art as Tie-2 ligand. (U.S. Pat. No. 5,643,755; Yancopoulos etal., 2000, Nature 407:242-248).

Antibodies and other peptide inhibitors that bind to Ang-2 are mentionedin, e.g., U.S. Pat. Nos. 6,166,185; 7,521,053; 7,205,275; 2006/0018909and 2006/0246071. There is a need in the art for novel Ang-2 modulatingagents, including Ang-2 antibodies, that can be used to treat diseasesand conditions caused by or exacerbated by angiogenesis.

BRIEF SUMMARY OF THE INVENTION

The present invention provides human antibodies that bind to humanAng-2. The present inventors, in view of various lines of evidence andinvestigation, have recognized a need for Ang-2 inhibitors which do notbind to or antagonize the related molecule Ang-1. For example, previousstudies have demonstrated or suggested a beneficial role for Ang-1 inhemostasis (see, e.g., Li et al., 2001, Thrombosis and Haemostasis85:191-374) and in protecting the adult vasculature against plasmaleakage (see, e.g., Thurston et al., 2000, Nature Medicine 6:460-463;Thurston et al., 1999, Science 286:2511-2514). Thus, the presentinventors recognized that, in certain anti-angiogenic therapeuticsituations, it may be beneficial to preserve Ang-1 activity.Accordingly, the present invention provides antibodies which bindspecifically to Ang-2 but do not substantially bind to Ang-1. Thepresent invention also includes antibodies that block the interactionbetween Ang-2 and its receptor Tie-2 but do not substantially block theinteraction between Ang-1 and Tie-2. The antibodies of the invention areuseful, inter alia, for inhibiting the angiogenesis-promoting activitiesof Ang-2 and for treating diseases and disorders caused by or related tothe process of angiogenesis.

The antibodies of the invention can be full-length (for example, an IgG1or IgG4 antibody) or may comprise only an antigen-binding portion (forexample, a Fab, F(ab′)₂ or scFv fragment), and may be modified to affectfunctionality, e.g., to eliminate residual effector functions.

In one embodiment, the invention comprises an antibody orantigen-binding fragment of an antibody comprising a heavy chainvariable region (HCVR) having an amino acid sequence selected from thegroup consisting of SEQ ID NO: 2, 18, 22, 26, 42, 46, 50, 66, 70, 74,90, 94, 98, 114, 118, 122, 138, 142, 146, 162, 166, 170, 186, 190, 194,210, 214, 218, 234, 238, 242, 258, 262, 266, 282, 286, 290, 306, 310,314, 330, 334, 338, 354, 358, 362, 378, 382, 386, 402, 406, 410, 426,430, 434, 450, 454, 458, 474, 478, 482, 498, 502, 506, 514, and 516, ora substantially similar sequence thereof having at least 90%, at least95%, at least 98% or at least 99% sequence identity. In one embodiment,the antibody or antigen-binding portion of an antibody comprises a HCVRhaving an amino acid sequence selected from the group consisting of SEQID NO: 18, 42, 66, 162, 210, 266, and 434.

In one embodiment, the invention comprises an antibody orantigen-binding fragment of an antibody comprising a light chainvariable region (LCVR) having an amino acid sequence selected from thegroup consisting of SEQ ID NO: 10, 20, 24, 34, 44, 48, 58, 68, 72, 82,92, 96, 106, 116, 120, 130, 140, 144, 154, 164, 168, 178, 188, 192, 202,212, 216, 226, 236, 240, 250, 260, 264, 274, 284, 288, 298, 308, 312,322, 332, 336, 346, 356, 360, 370, 380, 384, 394, 404, 408, 418, 428,432, 442, 452, 456, 466, 476, 480, 490, 500, and 504, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity. In one embodiment, the antibody orantigen-binding portion of an antibody comprises a LCVR having an aminoacid sequence selected from the group consisting of SEQ ID NO: 20, 44,68, 164, 212, 274, and 442.

In specific embodiments, the antibody or antigen-binding fragmentthereof comprises a HCVR and LCVR (HCVR/LCVR) amino acid sequence pairselected from the group consisting of SEQ ID NO: 2/10, 18/20, 22/24,26/34, 42/44, 46/48, 50/58, 66/68, 70/72, 74/82, 90/92, 94/96, 98/106,114/116, 118/120, 122/130, 138/140, 142/144, 146/154, 162/164, 166/168,170/178, 186/188, 190/192, 194/202, 210/212, 214/216, 218/226, 234/236,238/240, 242/250, 258/260, 262/264, 266/274, 282/284, 286/288, 290/298,306/308, 310/312, 314/322, 330/332, 334/336, 338/346, 354/356, 358/360,362/370, 378/380, 382/384, 386/394, 402/404, 406/408, 410/418, 426/428,430/432, 434/442, 450/452, 454/456, 458/466, 474/476, 478/480, 482/490,498/500, and 502/504. In one embodiment, the antibody or fragmentthereof comprises a HCVR and LCVR selected from the amino acid sequencepairs of SEQ ID NO: 18/20, 42/44, 66/68, 162/164, 210/212, 266/274, and434/442.

In a next aspect, the invention provides an antibody or antigen-bindingfragment of an antibody comprising a heavy chain CDR3 (HCDR3) domainhaving an amino acid sequence selected from the group consisting of SEQID NO: 8, 32, 56, 80, 104, 128, 152, 176, 200, 224, 248, 272, 296, 320,344, 368, 392, 416, 440, 464, 488, and 512, or a substantially similarsequence thereof having at least 90%, at least 95%, at least 98% or atleast 99% sequence identity; and a light chain CDR3 (LCDR3) domainselected from the group consisting of SEQ ID NO: 16, 40, 64, 88, 112,136, 160, 184, 208, 232, 256, 280, 304, 328, 352, 376, 400, 424, 448,472, and 496, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity.

In certain embodiments, the antibody or antigen-binding portion of anantibody comprises a HCDR3/LCDR3 amino acid sequence pair selected fromthe group consisting of SEQ ID NO: 8/16, 32/40, 56/64, 80/88, 104/112,128/136, 152/160, 176/184, 200/208, 224/232, 248/256, 272/280, 296/304,320/328, 344/352, 368/376, 392/400, 416/424, 440/448, 464/472, and488/496. In one embodiment, the antibody or antigen-binding portion ofan antibody comprises a HCDR3/LCDR3 amino acid sequence pair selectedfrom the group consisting of SEQ ID NO: 8/16, 32/40, 56/64, 152/160,200/208, 272/280, and 440/448. Non-limiting examples of anti-Ang-2antibodies having these HCDR3/LCDR3 pairs are the antibodies designatedH1H685, H1H690, H1H691, H1H696, H1H706, H1M724, and H2M744,respectively.

In a further embodiment, the invention comprises an antibody or fragmentthereof further comprising a HCDR1 domain having an amino acid sequenceselected from the group consisting of SEQ ID NO: 4, 28, 52, 76, 100,124, 148, 172, 196, 220, 244, 268, 292, 316, 340, 364, 388, 412, 436,460, 484, and 508, or a substantially similar sequence thereof having atleast 90%, at least 95%, at least 98% or at least 99% sequence identity;a heavy chain CDR2 (HCDR2) domain having an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 6, 30, 54, 78, 102, 126, 150,174, 198, 222, 246, 270, 294, 318, 342, 366, 390, 414, 438, 462, 486,and 510, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identity; LCDR1domain having an amino acid sequence selected from the group consistingof SEQ ID NO: 12, 36, 60, 84, 108, 132, 156, 180, 204, 228, 252, 276,300, 324, 348, 372, 396, 420, 444, 468, and 492, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity; and a LCDR2 domain having an aminoacid sequence selected from the group consisting of SEQ ID NO: 14, 38,62, 86, 110, 134, 158, 182, 206, 230, 254, 278, 302, 326, 350, 374, 398,422, 446, 470, and 494, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity.

Certain non-limiting, exemplary antibodies and antigen-binding fragmentsof the invention comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3domains, respectively, selected from the group consisting of: (i) SEQ IDNO: 4, 6, 8, 12, 14 and 16 (e.g., H1H685); (ii) SEQ ID NO: 28, 30, 32,36, 38 and 40 (e.g., H1H690); (iii) SEQ ID NO: 52, 54, 56, 60, 62 and 64(e.g., H1H691); (iv) SEQ ID NO: 148, 150, 152, 156, 158 and 160 (e.g.,H1H696); (v) SEQ ID NO: 196, 198, 200, 204, 206 and 208 (e.g., H1H706);(vi) SEQ ID NO: 268, 270, 272, 276, 278 and 280 (e.g., H1M724); and(vii) SEQ ID NO: 436, 438, 440, 444, 446 and 448 (e.g., H2M744).

In a related embodiment, the invention comprises an antibody orantigen-binding fragment of an antibody which specifically binds Ang-2,wherein the antibody or fragment comprises the heavy and light chain CDRdomains (i.e., CDR1, CDR2 and CDR3) contained within heavy and lightchain variable domain sequences selected from the group consisting ofSEQ ID NO: 2/10, 18/20, 22/24, 26/34, 42/44, 46/48, 50/58, 66/68, 70/72,74/82, 90/92, 94/96, 98/106, 114/116, 118/120, 122/130, 138/140,142/144, 146/154, 162/164, 166/168, 170/178, 186/188, 190/192, 194/202,210/212, 214/216, 218/226, 234/236, 238/240, 242/250, 258/260, 262/264,266/274, 282/284, 286/288, 290/298, 306/308, 310/312, 314/322, 330/332,334/336, 338/346, 354/356, 358/360, 362/370, 378/380, 382/384, 386/394,402/404, 406/408, 410/418, 426/428, 430/432, 434/442, 450/452, 454/456,458/466, 474/476, 478/480, 482/490, 498/500, and 502/504. In oneembodiment, the antibody or fragment thereof comprises the CDR sequencescontained within HCVR and LCVR selected from the amino acid sequencepairs of SEQ ID NO: 18/20, 42/44, 66/68, 162/164, 210/212, 266/274, and434/442.

In another aspect, the invention provides nucleic acid moleculesencoding anti-Ang-2 antibodies or fragments thereof. Recombinantexpression vectors carrying the nucleic acids of the invention, and hostcells into which such vectors have been introduced, are also encompassedby the invention, as are methods of producing the antibodies byculturing the host cells under conditions permitting production of theantibodies, and recovering the antibodies produced.

In one embodiment, the invention provides an antibody or fragmentthereof comprising a HCVR encoded by a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO: 1, 17, 21, 25, 41, 45, 49, 65,69, 73, 89, 93, 97, 113, 117, 121, 137, 141, 145, 161, 165, 169, 185,189, 193, 209, 213, 217, 233, 237, 241, 257, 261, 265, 281, 285, 289,305, 309, 313, 329, 333, 337, 353, 357, 361, 377, 381, 385, 401, 405,409, 425, 429, 433, 449, 453, 457, 473, 477, 481, 497, 501, 505, 513,and 515, or a substantially identical sequence having at least 90%, atleast 95%, at least 98%, or at least 99% identity thereto. In oneembodiment, the antibody or fragment thereof comprises a HCVR encoded bya nucleic acid sequence selected from the group consisting of SEQ ID NO:17, 41, 65, 161, 209, 265, and 433.

In one embodiment, the invention provides an antibody or fragmentthereof comprising a LCVR encoded by a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO: 9, 19, 23, 33, 43, 47, 57, 67,71, 81, 91, 95, 105, 115, 119, 129, 139, 143, 153, 163, 167, 177, 187,191, 201, 211, 215, 225, 235, 239, 249, 259, 263, 273, 283, 287, 297,307, 311, 321, 331, 335, 345, 355, 359, 369, 379, 383, 393, 403, 407,417, 427, 431, 441, 451, 455, 465, 475, 479, 489, 499, and 503, or asubstantially identical sequence having at least 90%, at least 95%, atleast 98%, or at least 99% identity thereto. In one embodiment, theantibody or fragment thereof comprises a LCVR encoded by a nucleic acidsequence selected from the group consisting of SEQ ID NO: 19, 43, 67,163, 211, 273, and 441.

In one embodiment, the invention provides an antibody or antigen-bindingfragment of an antibody comprising a HCDR3 domain encoded by anucleotide sequence selected from the group consisting of SEQ ID NO: 7,31, 55, 79, 103, 127, 151, 175, 199, 223, 247, 271, 295, 319, 343, 367,391, 415, 439, 463, 487, and 511, or a substantially identical sequencehaving at least 90%, at least 95%, at least 98%, or at least 99%identity thereto; and a LCDR3 domain encoded by a nucleotide sequenceselected from the group consisting of SEQ ID NO: 15, 39, 63, 87, 111,135, 159, 183, 207, 231, 255, 279, 303, 327, 351, 375, 399, 423, 447,471, and 495, or a substantially identical sequence having at least 90%,at least 95%, at least 98%, or at least 99% identity thereto. In oneembodiment, the antibody or fragment thereof comprises HCDR3 and LCDR3sequences encoded by the nucleic acid sequence pairs selected from thegroup consisting of SEQ ID NO: 7/15, 31/39, 55/63, 151/159, 199/207,271/279, and 439/447.

In a further embodiment, the antibody or fragment thereof furthercomprises: a HCDR1 domain encoded by a nucleotide sequence selected fromthe group consisting of SEQ ID NO: 3, 27, 51, 75, 99, 123, 147, 171,195, 219, 243, 267, 291, 315, 339, 363, 387, 411, 435, 459, 483, and507, or a substantially identical sequence having at least 90%, at least95%, at least 98%, or at least 99% identity thereto; a HCDR2 domainencoded by a nucleotide sequence selected from the group consisting ofSEQ ID NO: 5, 29, 53, 77, 101, 125, 149, 173, 197, 221, 245, 269, 293,317, 341, 365, 389, 413, 437, 461, 485, and 509, or a substantiallyidentical sequence having at least 90%, at least 95%, at least 98%, orat least 99% identity thereto; a LCDR1 domain encoded by a nucleotidesequence selected from the group consisting of SEQ ID NO: 11, 35, 59,83, 107, 131, 155, 179, 203, 227, 251, 275, 299, 323, 347, 371, 395,419, 443, 467, and 491, or a substantially identical sequence having atleast 90%, at least 95%, at least 98%, or at least 99% identity thereto;and a LCDR2 domain encoded by a nucleotide sequence selected from thegroup consisting of SEQ ID NO: 13, 37, 61, 85, 109, 133, 157, 181, 205,229, 253, 277, 301, 325, 349, 373, 397, 421, 445, 469, and 493, or asubstantially identical sequence having at least 90%, at least 95%, atleast 98%, or at least 99% identity thereto.

In one embodiment, the antibody or fragment thereof comprises the heavyand light chain CDR sequences encoded by the nucleic acid sequences ofSEQ ID NO: 17 and 19; SEQ ID NO: 41 and 43; SEQ ID NO: 65 and 67; SEQ IDNO: 161 and 163; SEQ ID NO: 209 and 211; SEQ ID NO: 265 and 273; or SEQID NO: 433 and 441.

The invention encompasses anti-Ang-2 antibodies having a modifiedglycosylation pattern. In some applications, modification to removeundesirable glycosylation sites may be useful. For example, the presentinvention encompasses modified versions of any antibody set forth hereinwherein the modified version lacks a fucose moiety present on theoligosaccharide chain, for example, to increase antibody dependentcellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC277:26733). In other applications, modification of galactosylation canbe made in order to modify complement dependent cytotoxicity (CDC).

In another aspect, the invention provides a pharmaceutical compositioncomprising a recombinant human antibody or fragment thereof whichspecifically binds Ang-2 and a pharmaceutically acceptable carrier ordiluent. In a related aspect, the invention features a composition whichis a combination of an Ang-2 inhibitor and a second therapeutic agent.In one embodiment, the Ang-2 inhibitor is an antibody or fragmentthereof. In one embodiment, the second therapeutic agent is any agentthat is advantageously combined with an Ang-2 inhibitor. Exemplaryagents that may be advantageously combined with an Ang-2 inhibitorinclude, without limitation, any agent that inhibits or reducesangiogenesis, other cancer therapeutic agents, anti-inflammatory agents,cytokine inhibitors, growth factor inhibitors, anti-hematopoieticfactors, non-steroidal anti-inflammatory drugs (NSAIDs), antiviralagents, and antibiotics.

In yet another aspect, the invention provides methods for inhibitingAng-2 activity using the anti-Ang-2 antibody or antigen-binding portionof the antibody of the invention, wherein the therapeutic methodscomprise administering a therapeutically effective amount of apharmaceutical composition comprising an antibody or antigen-bindingfragment of an antibody of the invention. The disorder treated is anydisease or condition which is improved, ameliorated, inhibited orprevented by removal, inhibition or reduction of Ang-2 activity.Preferably, the anti-Ang-2 antibody or antibody fragment of theinvention is useful to treat any disease or condition caused by,associated with, or perpetuated by the process of angiogenesis. Incertain embodiments of the invention, the anti-Ang-2 antibodies orantigen-binding portions thereof are useful for the treatment of cancer.In the context of cancer therapies, the anti-Ang-2 antibodies of theinvention or antigen-binding portions thereof can be administered aloneor in combination with other anti-cancer therapeutic antibodies,chemotherapeutic agents and/or radiation therapy. In other embodimentsof the present invention, the anti-Ang-2 antibodies or antigen-bindingfragments thereof are useful for the treatment of one or more eyedisorders, e.g., age-related macular degeneration, diabetic retinopathy,etc., and/or one or more inflammatory or infectious diseases.

Other embodiments will become apparent from a review of the ensuingdetailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an alignment of the last 88 C-terminal amino acids of humanAng-2 (residues 409 to 496 of SEQ ID NO:518) with the correspondingamino acid sequence of human Ang-1 (SEQ ID NO:531). Residues that differbetween hAng-1 and hAng-2 are indicated by white text and black shading.Asterisks (*) indicate the amino acids of hAng-2 which were shown tointeract with human Tie-2 by crystal structure analysis. See Barton etal., Nat. Struct. Mol. Biol. 13:524-532 (2006). Triangles (▴) indicatethe Tie-2-interacting amino acid positions that differ between hAng-2and hAng-1.

FIG. 2 (Panels A-C) depict the results of Western blots which illustratethe extent to which Ang-2 binding molecules inhibit, or fail to inhibit,Ang-1-induced Tie-2 phosphorylation.

FIG. 3 is a summary of the Ang-2FD-mFc point mutant binding experimentof Example 13, showing the amino acid changes which resulted in greaterthan a five-fold reduction in T1/2 of dissociation (depicted by solidcircles ●) relative to wild-type for the various antibodies andpeptibodies tested.

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described. Allpatents, applications and non-patent publications mentioned in thisspecification are incorporated herein by reference in their entireties.

Definitions

As used herein, the term “angiopoietin-2” or “Ang-2”, unless specifiedas being from a non-human species (e.g., “mouse Ang-2,” “monkey Ang-2,”etc.), refers to human Ang-2 or a biologically active fragment thereof(e.g., a fragment of the Ang-2 protein which is capable of inducingangiogenesis in vitro or in vivo). Human Ang-2 is encoded by the nucleicacid sequence shown in SEQ ID NO:517 and has the amino acid sequence ofSEQ ID NO:518. The amino acid sequences of mouse and monkey Ang-2proteins are available from the NCBI protein sequence database underAccession Nos. NP_(—)031452 and BAE89705.1, respectively.

The term “angiopoietin-1” or “Ang-1”, unless specified as being from anon-human species (e.g., “mouse Ang-1,” “monkey Ang-1,” etc.), refers tohuman Ang-1 or a biologically active fragment thereof. Human Ang-1 hasthe amino acid sequence as set forth in the NCBI protein sequencedatabase under Accession No. AAB50557. The term “Tie-2” (also referredto in the art as “TEK”) unless specified as being from a non-humanspecies (e.g., “mouse Tie-2,” “monkey Tie-2,” etc.), refers to humanTie-2 or a biologically active fragment thereof. Human Tie-2 has theamino acid sequence as set forth in the NCBI protein sequence databaseunder Accession No. AAA61130.

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprising four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds,as well as multimers thereof (e.g., IgM). Each heavy chain comprises aheavy chain variable region (abbreviated herein as HCVR or V_(H)) and aheavy chain constant region. The heavy chain constant region comprisesthree domains, C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises alight chain variable region (abbreviated herein as LCVR or V_(L)) and alight chain constant region. The light chain constant region comprisesone domain (C_(L)1). The V_(H) and V_(L) regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDRs), interspersed with regions that are moreconserved, termed framework regions (FR). Each V_(H) and V_(L) iscomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. In different embodiments of the invention, the FRs of theanti-Ang-2 antibody (or antigen-binding portion thereof) may beidentical to the human germline sequences, or may be naturally orartificially modified. An amino acid consensus sequence may be definedbased on a side-by-side analysis of two or more CDRs.

The term “antibody,” as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)₂ fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR)). Other engineered molecules,such as diabodies, triabodies, tetrabodies and minibodies, are alsoencompassed within the expression “antigen-binding fragment,” as usedherein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDRwhich is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (V)V_(H)-C_(H)1-C_(H)2-C_(H)3; (vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L);(viii) V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (X) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemonospecific or multispecific (e.g., bispecific). A multispecificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multispecific antibody format, including theexemplary bispecific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques available in the art.

The constant region of an antibody is important in the ability of anantibody to fix complement and mediate cell-dependent cytotoxicity.Thus, the isotype of an antibody may be selected on the basis of whetherit is desirable for the antibody to mediate cytotoxicity.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell (describedfurther below), antibodies isolated from a recombinant, combinatorialhuman antibody library (described further below), antibodies isolatedfrom an animal (e.g., a mouse) that is transgenic for humanimmunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res.20:6287-6295) or antibodies prepared, expressed, created or isolated byany other means that involves splicing of human immunoglobulin genesequences to other DNA sequences. Such recombinant human antibodies havevariable and constant regions derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies are subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

Human antibodies can exist in two forms that are associated with hingeheterogeneity. In one form, an immunoglobulin molecule comprises astable four chain construct of approximately 150-160 kDa in which thedimers are held together by an interchain heavy chain disulfide bond. Ina second form, the dimers are not linked via inter-chain disulfide bondsand a molecule of about 75-80 kDa is formed composed of a covalentlycoupled light and heavy chain (half-antibody). These forms have beenextremely difficult to separate, even after affinity purification.

The frequency of appearance of the second form in various intact IgGisotypes is due to, but not limited to, structural differencesassociated with the hinge region isotype of the antibody. A single aminoacid substitution in the hinge region of the human IgG4 hinge cansignificantly reduce the appearance of the second form (Angal et al.(1993) Molecular Immunology 30:105) to levels typically observed using ahuman IgG1 hinge. The instant invention encompasses antibodies havingone or more mutations in the hinge, C_(H)2 or C_(H)3 region which may bedesirable, for example, in production, to improve the yield of thedesired antibody form.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds human Ang-2 or a human Ang-2 fragment is substantially free ofantibodies that specifically bind antigens other than human Ang-2). Theterm “specifically binds,” or the like, means that an antibody orantigen-binding fragment thereof forms a complex with an antigen that isrelatively stable under physiologic conditions. Specific binding can becharacterized by a K_(D) of about 1×10⁻⁸ M or less. Methods fordetermining whether two molecules specifically bind are well known inthe art and include, for example, equilibrium dialysis, surface plasmonresonance, and the like. An isolated antibody that specifically bindshuman Ang-2 may, however, have cross-reactivity to other antigens, suchas Ang-2 molecules from other species. Moreover, an isolated antibodymay be substantially free of other cellular material and/or chemicals.

A “neutralizing” or “blocking” antibody, as used herein, is intended torefer to an antibody whose binding to Ang-2 blocks the interactionbetween Ang-2 and its receptor (Tie-2) and/or results in inhibition ofat least one biological function of Ang-2. The inhibition caused by anAng-2 neutralizing or blocking antibody need not be complete so long asit is detectable using an appropriate assay. Exemplary assays fordetecting Ang-2 inhibition are described elsewhere herein.

The fully-human anti-Ang-2 antibodies disclosed herein may comprise oneor more amino acid substitutions, insertions and/or deletions in theframework and/or CDR regions of the heavy and light chain variabledomains as compared to the corresponding germline sequences. Suchmutations can be readily ascertained by comparing the amino acidsequences disclosed herein to germline sequences available from, forexample, public antibody sequence databases. The present inventionincludes antibodies, and antigen-binding fragments thereof, which arederived from any of the amino acid sequences disclosed herein, whereinone or more amino acids within one or more framework and/or CDR regionsare back-mutated to the corresponding germline residue(s) or to aconservative amino acid substitution (natural or non-natural) of thecorresponding germline residue(s) (such sequence changes are referred toherein as “germline back-mutations”). A person of ordinary skill in theart, starting with the heavy and light chain variable region sequencesdisclosed herein, can easily produce numerous antibodies andantigen-binding fragments which comprise one or more individual germlineback-mutations or combinations thereof. In certain embodiments, all ofthe framework and/or CDR residues within the V_(H) and/or V_(L) domainsare mutated back to the germline sequence. In other embodiments, onlycertain residues are mutated back to the germline sequence, e.g., onlythe mutated residues found within the first 8 amino acids of FR1 orwithin the last 8 amino acids of FR4, or only the mutated residues foundwithin CDR1, CDR2 or CDR3. Furthermore, the antibodies of the presentinvention may contain any combination of two or more germlineback-mutations within the framework and/or CDR regions, i.e., whereincertain individual residues are mutated back to the germline sequencewhile certain other residues that differ from the germline sequence aremaintained. Once obtained, antibodies and antigen-binding fragments thatcontain one or more germline back-mutations can be easily tested for oneor more desired property such as, improved binding specificity,increased binding affinity, improved or enhanced antagonistic oragonistic biological properties (as the case may be), reducedimmunogenicity, etc. Antibodies and antigen-binding fragments obtainedin this general manner are encompassed within the present invention.

The present invention also includes anti-Ang-2 antibodies comprisingvariants of any of the HCVR, LCVR, and/or CDR amino acid sequencesdisclosed herein having one or more conservative substitutions. Forexample, the present invention includes anti-Ang-2 antibodies havingHCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acidsubstitutions relative to any of the HCVR, LCVR, and/or CDR amino acidsequences disclosed herein. In one embodiment, the antibody comprises anHCVR having the amino acid sequence of SEQ ID NO:18 with 8 or fewerconservative amino acid substitutions. In another embodiment, theantibody comprises an HCVR having the amino acid sequence of SEQ IDNO:18 with 6 or fewer conservative amino acid substitutions. In anotherembodiment, the antibody comprises an HCVR having the amino acidsequence of SEQ ID NO:18 with 4 or fewer conservative amino acidsubstitutions. In another embodiment, the antibody comprises an HCVRhaving the amino acid sequence of SEQ ID NO:18 with 2 or fewerconservative amino acid substitutions. In one embodiment, the antibodycomprises an LCVR having the amino acid sequence of SEQ ID NO:20 with 8or fewer conservative amino acid substitutions. In another embodiment,the antibody comprises an LCVR having the amino acid sequence of SEQ IDNO:20 with 6 or fewer conservative amino acid substitutions. In anotherembodiment, the antibody comprises an LCVR having the amino acidsequence of SEQ ID NO:20 with 4 or fewer conservative amino acidsubstitutions. In another embodiment, the antibody comprises an LCVRhaving the amino acid sequence of SEQ ID NO:20 with 2 or fewerconservative amino acid substitutions.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-timeinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore™ system(Biacore Life Sciences division of GE Healthcare, Piscataway, N.J.).

The term “K_(D)”, as used herein, is intended to refer to theequilibrium dissociation constant of a particular antibody-antigeninteraction.

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen binding site in the variable region of anantibody molecule known as a paratope. A single antigen may have morethan one epitope. Thus, different antibodies may bind to different areason an antigen and may have different biological effects. Epitopes may beeither conformational or linear. A conformational epitope is produced byspatially juxtaposed amino acids from different segments of the linearpolypeptide chain. A linear epitope is one produced by adjacent aminoacid residues in a polypeptide chain. In certain circumstance, anepitope may include moieties of saccharides, phosphoryl groups, orsulfonyl groups on the antigen.

The term “substantial identity” or “substantially identical,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 95%, and more preferablyat least about 96%, 97%, 98% or 99% of the nucleotide bases, as measuredby any well-known algorithm of sequence identity, such as FASTA, BLASTor Gap, as discussed below. A nucleic acid molecule having substantialidentity to a reference nucleic acid molecule may, in certain instances,encode a polypeptide having the same or substantially similar amino acidsequence as the polypeptide encoded by the reference nucleic acidmolecule.

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 95% sequence identity, even more preferably atleast 98% or 99% sequence identity. Preferably, residue positions whichare not identical differ by conservative amino acid substitutions. A“conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chain(R group) with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein. Incases where two or more amino acid sequences differ from each other byconservative substitutions, the percent sequence identity or degree ofsimilarity may be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment arewell-known to those of skill in the art. See, e.g., Pearson (1994)Methods Mol. Biol. 24: 307-331. Examples of groups of amino acids thathave side chains with similar chemical properties include (1) aliphaticside chains: glycine, alanine, valine, leucine and isoleucine; (2)aliphatic-hydroxyl side chains: serine and threonine; (3)amide-containing side chains: asparagine and glutamine; (4) aromaticside chains: phenylalanine, tyrosine, and tryptophan; (5) basic sidechains: lysine, arginine, and histidine; (6) acidic side chains:aspartate and glutamate, and (7) sulfur-containing side chains arecysteine and methionine. Preferred conservative amino acids substitutiongroups are: valine-leucine-isoleucine, phenylalanine-tyrosine,lysine-arginine, alanine-valine, glutamate-aspartate, andasparagine-glutamine. Alternatively, a conservative replacement is anychange having a positive value in the PAM250 log-likelihood matrixdisclosed in Gonnet et al. (1992) Science 256: 1443-1445. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG software contains programs such as Gap and Bestfitwhich can be used with default parameters to determine sequence homologyor sequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson (2000) supra). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially BLASTP or TBLASTN, using default parameters. See, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al.(1997) Nucleic Acids Res. 25:3389-402.

Preparation of Human Antibodies

Methods for generating monoclonal antibodies, including fully humanmonoclonal antibodies are known in the art. Any such known methods canbe used in the context of the present invention to make human antibodiesthat specifically bind to human Ang-2 and which possess one or more ofthe antigen-binding and/or functional characteristics of any of theexemplary anti-Ang-2 antibodies disclosed herein.

Using VELOCIMMUNE™ technology or any other known method for generatingmonoclonal antibodies, high affinity chimeric antibodies to Ang-2 areinitially isolated having a human variable region and a mouse constantregion. As in the experimental section below, the antibodies arecharacterized and selected for desirable characteristics, includingaffinity, selectivity, epitope, etc. The mouse constant regions arereplaced with a desired human constant region to generate the fullyhuman antibody of the invention, for example wild-type or modified IgG1or IgG4. While the constant region selected may vary according tospecific use, high affinity antigen-binding and target specificitycharacteristics reside in the variable region.

Bioequivalents

The anti-Ang-2 antibodies and antibody fragments of the presentinvention encompass proteins having amino acid sequences that vary fromthose of the described antibodies, but that retain the ability to bindhuman Ang-2. Such variant antibodies and antibody fragments comprise oneor more additions, deletions, or substitutions of amino acids whencompared to parent sequence, but exhibit biological activity that isessentially equivalent to that of the described antibodies. Likewise,the anti-Ang-2 antibody-encoding DNA sequences of the present inventionencompass sequences that comprise one or more additions, deletions, orsubstitutions of nucleotides when compared to the disclosed sequence,but that encode an anti-Ang-2 antibody or antibody fragment that isessentially bioequivalent to an anti-Ang-2 antibody or antibody fragmentof the invention. Examples of such variant amino acid and DNA sequencesare discussed above.

Two antigen-binding proteins, or antibodies, are consideredbioequivalent if, for example, they are pharmaceutical equivalents orpharmaceutical alternatives whose rate and extent of absorption do notshow a significant difference when administered at the same molar doseunder similar experimental conditions, either single does or multipledose. Some antibodies will be considered equivalents or pharmaceuticalalternatives if they are equivalent in the extent of their absorptionbut not in their rate of absorption and yet may be consideredbioequivalent because such differences in the rate of absorption areintentional and are reflected in the labeling, are not essential to theattainment of effective body drug concentrations on, e.g., chronic use,and are considered medically insignificant for the particular drugproduct studied.

In one embodiment, two antigen-binding proteins are bioequivalent ifthere are no clinically meaningful differences in their safety, purity,and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if apatient can be switched one or more times between the reference productand the biological product without an expected increase in the risk ofadverse effects, including a clinically significant change inimmunogenicity, or diminished effectiveness, as compared to continuedtherapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent ifthey both act by a common mechanism or mechanisms of action for thecondition or conditions of use, to the extent that such mechanisms areknown.

Bioequivalence may be demonstrated by in vivo and in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the antibody or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the antibody (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of an antibody.

Bioequivalent variants of anti-Ang-2 antibodies of the invention may beconstructed by, for example, making various substitutions of residues orsequences or deleting terminal or internal residues or sequences notneeded for biological activity. For example, cysteine residues notessential for biological activity can be deleted or replaced with otheramino acids to prevent formation of unnecessary or incorrectintramolecular disulfide bridges upon renaturation.

Biological and Therapeutic Characteristics of the Antibodies

In general, the antibodies of the instant invention bind to human Ang-2with a K_(D) of less than 100 pM, typically with a K_(D) of less than 50pM, and in certain embodiments, with a K_(D) of less than 40 pM, whenmeasured by binding to antigen either immobilized on solid phase or insolution phase.

In addition, certain exemplary anti-Ang-2 antibodies of the inventionmay exhibit one or more of the following characteristics: (1) ability tobind to human Ang-2 but not to mouse Ang-2; (2) ability to bind to humanAng-2 and to mouse Ang-2; (3) ability to bind to human Ang-2 but not tohuman Ang-1, -3 or -4; (4) ability to bind to human Ang-2 but not tomouse Ang-1, -3 or -4; (5) ability to bind to human Ang-2 and to humanAng-1, -3 or -4; (6) ability to bind to human Ang-2 and to mouse Ang-1,-3 or -4; (7) ability to block binding of human Ang-2 to human Tie-2;(8) ability to block binding of human Ang-2 to mouse Tie-2; (9) abilityto block binding of mouse Ang-2 to human Tie-2; (10) ability to blockbinding of mouse Ang-2 to mouse Tie-2; (11) ability to block binding ofhuman Ang-1 to human Tie-2; (12) ability to block binding of human Ang-1to mouse Tie-2; (13) ability to block binding of mouse Ang-1 to humanTie-2; (14) ability to block binding of mouse Ang-1 to mouse Tie-2; (15)ability to inhibit human Ang-2-induced phosphorylation of human Tie-2;(16) ability to inhibit human Ang-2-induced phosphorylation of mouseTie-2; (17) ability to inhibit mouse Ang-2-induced phosphorylation ofhuman Tie-2; (18) ability to inhibit mouse Ang-2 induced phosphorylationof mouse Tie-2; (19) ability to inhibit human Ang-1-inducedphosphorylation of human Tie-2; (20) ability to inhibit humanAng-1-induced phosphorylation of mouse Tie-2; (21) ability to inhibitmouse Ang-1-induced phosphorylation of human Tie-2; (22) ability toinhibit mouse-Ang-1-induced phosphorylation of mouse Tie-2; (23) abilityto inhibit in vivo angiogenesis in an experimental model (e.g.,angiogenesis induced by a Matrigel plug containing MCF-7 cells implantedsubcutaneously into nude mice); and/or (24) ability to inhibit ordecrease tumor volume in a mouse xenograft model.

The present invention also includes antibodies that bind with highaffinity to a construct comprising the Ang-2 fibronectin-like domain butlacking the Ang-2 N-terminal coiled-coil domain (such constructs arereferred to herein as “Ang-2FD”). Exemplary Ang-2FD constructs includehuman Ang-2FD (SEQ ID NO:519), mouse Ang-2FD (SEQ ID NO:520), and monkeyAng-2FD (SEQ ID NO:521). The human, mouse and monkey Ang-2FD constructsmay be monomeric or dimeric. Ang-2FD constructs may also include othernon-Ang-2 amino acid sequences such as a human or mouse Fc domain linkedto the Ang-2FD molecules. Another exemplary Ang-2FD construct isreferred to herein as “h8A2” (or human “bow-Ang2”) which is a tetramerof human Ang-2 fibrinogen-like domains associated with one another via ahuman or mouse Fc domain to form a bow-tie-like configuration.Typically, hBA2 consists of two Ang-2 dimers, wherein each Ang-2 dimercontains two Ang-2 fibronectin-like domains connected to one another viaan Fc domain. Exemplary hBA2 components include the polypeptidesdesignated hBA2-hIgG1 (SEQ ID NO:522) and hBA2-mIgG2a (SEQ ID NO:523).Unexpectedly, certain anti-Ang-2 antibodies of the present inventionwere found to bind to Ang-2FD constructs with much higher affinitiesthan an known Ang-2 control antibody (see Examples set forth herein).

High affinity binding, in the context of anti-Ang-2 antibody binding toa human or mouse dimeric Ang-2FD construct, means that the anti-Ang-2antibody binds the human or mouse dimeric Ang-2FD with a K_(D) of lessthan 300 pM. For example, anti-Ang-2 antibodies that bind with highaffinity to human or mouse dimeric Ang-2FD include antibodies that bindto human or mouse dimeric Ang-2-FD with a K_(D) of less than 300 pM,less than 250 pM, less than 200 pM, less than 190 pM, less than 180 pM,less than 170 pM, less than 160 pM, less than 150 pM, less than 140 pM,less than 130 pM, less than 120 pM, less than 110 pM, less than 100 pM,less than 90 pM, less than 80 pM, less than 70 pM, less than 60 pM orless than 50 pM, as measured at 25° C. in a surface Plasmon resonanceassay.

High affinity binding, in the context of anti-Ang-2 antibody binding toa monkey dimeric Ang-2FD construct, means that the anti-Ang-2 antibodybinds the monkey dimeric Ang-2FD with a K_(D) of less than 500 pM. Forexample, anti-Ang-2 antibodies that bind with high affinity to monkeydimeric Ang-2FD include antibodies that bind to monkey Ang-2-FD with aK_(D) of less than 500 pM, less than 450 pM, less than 400 pM, less than350 pM, less than 300 pM, less than 250 pM, less than 200 pM, less than190 pM, less than 180 pM, less than 170 pM, less than 160 pM, less than150 pM, less than 140 pM, less than 130 pM, less than 120 pM, less than110 pM, less than 100 pM, less than 90 pM, or less than 80 pM, asmeasured at 25° C. in a surface Plasmon resonance assay.

High affinity binding, in the context of anti-Ang-2 antibody binding toa human monomeric Ang-2FD construct, means that the anti-Ang-2 antibodybinds the human monomeric Ang-2FD with a K_(D) of less than 40 nM. Forexample, anti-Ang-2 antibodies that bind with high affinity to humanmonomeric Ang-2FD include antibodies that bind to human monomericAng-2-FD with a K_(D) of less than 40 nM, less than 30 nM, less than 25nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 9 nM,less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, lessthan 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.9nM, less than 0.8 nM, less than 0.7 nM, or less than 0.6 nM as measuredat 25° C. in a surface Plasmon resonance assay.

High affinity binding, in the context of anti-Ang-2 antibody binding toa hBA2 construct, means that the anti-Ang-2 antibody binds the hBA2 witha K_(D) of less than 80 pM. For example, anti-Ang-2 antibodies that bindwith high affinity to hBA2 include antibodies that bind to hBA2 with aK_(D) of less than 80 pM, less than 75 pM, less than 70 pM, less than 65pM, less than 60 pM, less than 55 pM, less than 50 pM, less than 45 pM,less than 40 pM, less than 35 pM, less than 30 pM, less than 25 pM, lessthan 20 pM, less than 18 pM, less than 16 pM, less than 14 pM, or lessthan 12 pM, as measured at 25° C. in a surface Plasmon resonance assay.

The present invention includes antibodies that bind Ang-2 but do notsubstantially bind Ang-1. As used herein, an antibody “does notsubstantially bind Ang-1” if the antibody, when tested for binding toAng-1 in a surface plasmon resonance assay in which the antibody iscaptured on a surface and full-length wild-type human Ang-1 at aconcentration of about 25 nM is injected over the captured antibodysurface at a flowrate of about 60 μl/min for about 3 minutes at 25° C.,exhibits a K_(D) of greater than about 1 nM, e.g., a K_(D) of greaterthan about 5 nM, greater than about 10 nM, greater than about 50 nM,greater than about 100 nM, greater than about 150 nM, greater than about200 nM, greater than about 250 nM, greater than about 300 nM, greaterthan about 350 nM, greater than about 400 nM, greater than about 450 nM,greater than about 500 nM, or more. (See, e.g., Example 4). In addition,an antibody “does not substantially bind Ang-1” if the antibody fails toexhibit any binding to Ang-1 when tested in such an assay or equivalentthereof.

The present invention also includes antibodies that block the binding ofAng-2 to Tie-2 but do not substantially block the binding of Ang-1 toTie-2. As used herein, an antibody “does not substantially block thebinding of Ang-1 to Tie-2” if, when the antibody is premixed with Ang-1antigen at a ratio of about 100:1 (antibody:antigen) and allowed toincubate at 25° C. for about 60 minutes and then the equilibratedmixture is tested for binding to Tie-2 by surface plasmon resonance overa Tie-2-coated surface (5 μl/min for 5 min. at 25° C.), the amount ofAng-1 bound to Tie-2 is at least 50% the amount of Ang-1 bound to Tie-2in the presence of an irrelevant control molecule. (See, e.g., Example6). For example, if the amount of Ang-1 bound to Tie-2 followingpreincubation with an antibody is at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, or about 100% the amount of Ang-1 that binds toTie-2 following preincubation with an irrelevant control molecule underthe above noted experimental conditions, then the antibody is deemed to“not substantially block the binding of Ang-1 to Tie-2.”

Moreover, the present invention includes antibodies that block orsubstantially attenuate a biological activity of Ang-2 (e.g.,Ang-2-mediated phosphorylation of Tie-2; Ang-2-induced angiogenesis;etc.) but do not block or substantially attenuate the correspondingbiological activity of Ang-1 (e.g., Ang-1-mediated phosphorylation ofTie-2; Ang-1-induced angiogenesis; etc). Assays and tests useful fordetermining whether an antibody satisfies one or more of thecharacteristics listed above will be readily known and easily practicedby persons of ordinary skill in the art and/or can be fully ascertainedfrom the present disclosure. For example, the experimental proceduresdetailed below can be used to determine whether a given antibody bindsor does not bind to Ang-2 and/or Ang-1; blocks or does not block bindingof Ang-2 and/or Ang-1 to Tie-2; inhibits or does not inhibit Ang-2-and/or Ang-1-mediated phosphorylation of Tie-2; etc.

Epitope Mapping and Related Technologies

To screen for antibodies that bind to a particular epitope (e.g., thosewhich block binding of IgE to its high affinity receptor), a routinecross-blocking assay such as that described “Antibodies,” Harlow andLane (Cold Spring Harbor Press, Cold Spring Harb., NY) can be performed.Other methods include alanine scanning mutants, peptide blots (Reineke(2004) Methods Mol Biol 248:443-63), or peptide cleavage analysis. Inaddition, methods such as epitope excision, epitope extraction andchemical modification of antigens can be employed (Tomer (2000) ProteinScience 9: 487-496).

The term “epitope” refers to a site on an antigen to which B and/or Tcells respond. B-cell epitopes can be formed both from contiguous aminoacids or noncontiguous amino acids juxtaposed by tertiary folding of aprotein. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents, whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation.

Modification-Assisted Profiling (MAP), also known as AntigenStructure-based Antibody Profiling (ASAP) is a method that categorizeslarge numbers of monoclonal antibodies (mAbs) directed against the sameantigen according to the similarities of the binding profile of eachantibody to chemically or enzymatically modified antigen surfaces (US2004/0101920). Each category may reflect a unique epitope eitherdistinctly different from or partially overlapping with epitoperepresented by another category. This technology allows rapid filteringof genetically identical antibodies, such that characterization can befocused on genetically distinct antibodies. When applied to hybridomascreening, MAP may facilitate identification of rare hybridoma clonesthat produce mAbs having the desired characteristics. MAP may be used tosort the anti-Ang-2 antibodies of the invention into groups ofantibodies binding different epitopes.

Anti-Ang-2 antibodies can bind to an epitope within the amino-terminalcoiled-coil domain or within the carboxy-terminal fibrinogen-like domain(“FD”). In preferred embodiments of the present invention, theanti-Ang-2 antibodies and antigen binding fragments thereof bind to anepitope within the FD.

The amino acids within the FD of Ang-2 that interact with Tie-2 havebeen ascertained from crystal structure analysis. See Barton et al.,Nat. Struct. Mol. Biol. 13:524-532 (May 2006). With regard to antibodiesthat block the binding of Ang-2 to Tie-2 but do not substantially blockbinding of Ang-1 to Tie-2 (e.g., H1H685P, see Examples 5 and 6 below),the epitope to which such antibodies bind may include one or more aminoacids of Ang-2 that (a) interact with Tie-2 and (b) are non-identical tothe corresponding amino acid in Ang-1. (See FIG. 1). Thus, the epitopeto which such Ang-2 preferential antibodies bind may include one or moreof the following amino acids of hAng-2 (SEQ ID NO:518): S-417; K-432;1-434; N-467; F-469; Y-475; or S-480. For example, the present inventorshave discovered that antibodies which interact with amino acids F-469,Y-475, and S-480 of Ang-2 (SEQ ID NO:518) preferentially interact withAng-2 over Ang-1, and this preferential binding may have therapeuticbenefits. Thus, the present invention includes anti-Ang-2 antibodieswhich specifically bind human angiopoietin-2 (hAng-2) but do notsubstantially bind hAng-1, wherein the antibodies bind an epitope onhAng-2 (SEQ ID NO:518) comprising amino acids F-469, Y-475, and S-480.Similarly, the present invention includes anti-Ang-2 antibodies whichblock the binding of hAng-2 to hTie-2 but do not substantially block thebinding of hAng-1 to hTie-2, wherein the antibodies bind an epitope onhAng-2 (SEQ ID NO:518) comprising amino acids F-469, Y-475, and S-480.

The present invention includes anti-Ang-2 antibodies that bind to thesame epitope as any of the specific exemplary antibodies describedherein (e.g., H1H685, H1H690, H1H691, H1H696, H1H706, H1M724 and/orH2M744). Likewise, the present invention also includes anti-Ang-2antibodies that compete for binding to Ang-2 with any of the specificexemplary antibodies described herein (e.g., H1H685, H1H690, H1H691,H1H696, H1H706, H1M724 and/or H2M744).

One can easily determine whether an antibody binds to the same epitopeas, or competes for binding with, a reference anti-Ang-2 antibody byusing routine methods known in the art. For example, to determine if atest antibody binds to the same epitope as a reference anti-Ang-2antibody of the invention, the reference antibody is allowed to bind toan Ang-2 protein or peptide under saturating conditions. Next, theability of a test antibody to bind to the Ang-2 molecule is assessed. Ifthe test antibody is able to bind to Ang-2 following saturation bindingwith the reference anti-Ang-2 antibody, it can be concluded that thetest antibody binds to a different epitope than the reference anti-Ang-2antibody. On the other hand, if the test antibody is not able to bind tothe Ang-2 molecule following saturation binding with the referenceanti-Ang-2 antibody, then the test antibody may bind to the same epitopeas the epitope bound by the reference anti-Ang-2 antibody of theinvention. Additional routine experimentation (e.g., peptide mutationand binding analyses) can then be carried out to confirm whether theobserved lack of binding of the test antibody is in fact due to bindingto the same epitope as the reference antibody or if steric blocking (oranother phenomenon) is responsible for the lack of observed binding.Experiments of this sort can be performed using ELISA, RIA, Biacore,flow cytometry or any other quantitative or qualitative antibody-bindingassay available in the art. In accordance with certain embodiments ofthe present invention, two antibodies bind to the same (or overlapping)epitope if, e.g., a 1-, 5-, 10-, 20- or 100-fold excess of one antibodyinhibits binding of the other by at least 50% but preferably 75%, 90% oreven 99% as measured in a competitive binding assay (see, e.g., Junghanset al., Cancer Res. 1990:50:1495-1502). Alternatively, two antibodiesare deemed to bind to the same epitope if essentially all amino acidmutations in the antigen that reduce or eliminate binding of oneantibody reduce or eliminate binding of the other. Two antibodies aredeemed to have “overlapping epitopes” if only a subset of the amino acidmutations that reduce or eliminate binding of one antibody reduce oreliminate binding of the other.

To determine if an antibody competes for binding with a referenceanti-Ang-2 antibody, the above-described binding methodology isperformed in two orientations: In a first orientation, the referenceantibody is allowed to bind to an Ang-2 molecule under saturatingconditions followed by assessment of binding of the test antibody to theAng-2 molecule. In a second orientation, the test antibody is allowed tobind to an Ang-2 molecule under saturating conditions followed byassessment of binding of the reference antibody to the Ang-2 molecule.If, in both orientations, only the first (saturating) antibody iscapable of binding to the Ang-2 molecule, then it is concluded that thetest antibody and the reference antibody compete for binding to Ang-2.As will be appreciated by a person of ordinary skill in the art, anantibody that competes for binding with a reference antibody may notnecessarily bind to the same epitope as the reference antibody, but maysterically block binding of the reference antibody by binding anoverlapping or adjacent epitope.

Species Selectivity and Species Cross-Reactivity

According to certain embodiments of the invention, the anti-Ang-2antibodies bind to human Ang-2 but not to Ang-2 from other species.Alternatively, the anti-Ang-2 antibodies of the invention, in certainembodiments, bind to human Ang-2 and to Ang-2 from one or more non-humanspecies. For example, the Ang-2 antibodies of the invention may bind tohuman Ang-2 and may bind or not bind, as the case may be, to one or moreof mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat,sheep, cow, horse, camel, cynomologous, marmoset, rhesus or chimpanzeeAng-2.

Immunoconjugates

The invention encompasses anti-Ang-2 monoclonal antibodies conjugated toa therapeutic moiety (“immunoconjugate”), such as a cytotoxin, achemotherapeutic drug, an immunosuppressant or a radioisotope. Cytotoxicagents include any agent that is detrimental to cells. Examples ofsuitable cytotoxic agents and chemotherapeutic agents for formingimmunoconjugates are known in the art, see for example, WO 05/103081).

Multispecific Antibodies

The antibodies of the present invention may be monospecific, bispecific,or multispecific. Multispecific mAbs may be specific for differentepitopes of one target polypeptide or may contain antigen-bindingdomains specific for more than one target polypeptide. See, e.g., Tuttet al. (1991) J. Immunol. 147:60-69. The anti-Ang-2 antibodies of thepresent invention, or portions thereof, can be linked to or co-expressedwith another functional molecule, e.g., another peptide or protein, toform a multispecific molecule. For example, an antibody or fragmentthereof can be functionally linked (e.g., by chemical coupling, geneticfusion, noncovalent association or otherwise) to one or more othermolecular entities, such as another antibody or antibody fragment, toproduce a bispecific or a multispecific antibody with a second bindingspecificity.

An exemplary bi-specific antibody format that can be used in the contextof the present invention involves the use of a first immunoglobulin (Ig)C_(H)3 domain and a second Ig C_(H)3 domain, wherein the first andsecond Ig C_(H)3 domains differ from one another by at least one aminoacid, and wherein at least one amino acid difference reduces binding ofthe bispecific antibody to Protein A as compared to a bi-specificantibody lacking the amino acid difference. In one embodiment, the firstIg C_(H)3 domain binds Protein A and the second Ig C_(H)3 domaincontains a mutation that reduces or abolishes Protein A binding such asan H95R modification (by IMGT exon numbering; H435R by EU numbering).The second C_(H)3 may further comprise a Y96F modification (by IMGT;Y436F by EU). Further modifications that may be found within the secondC_(H)3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E,L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU)in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q,and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422Iby EU) in the case of IgG4 antibodies. Variations on the bi-specificantibody format described above are contemplated within the scope of thepresent invention.

Therapeutic Formulation and Administration

The invention provides therapeutic compositions comprising theanti-Ang-2 antibodies or antigen-binding fragments thereof of thepresent invention. The therapeutic compositions in of the presentinvention may further comprise one or more pharmaceutically acceptablecarriers, excipients, and other agents that are incorporated intoformulations to provide improved transfer, delivery, tolerance, and thelike (herein collectively referred to as “pharmaceutically acceptablecarriers or diluents”). A multitude of appropriate formulations can befound in the formulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. See also Powell et al.“Compendium of excipients for parenteral formulations” PDA, 1998, JPharm Sci Technol 52:238-311.

The dose of antibody may vary depending upon the age and the size of asubject to be administered, target disease, conditions, route ofadministration, and the like. The preferred dose is typically calculatedaccording to body weight or body surface area. When an antibody of thepresent invention is used for treating a condition or disease associatedwith Ang-2 activity in an adult patient, it may be advantageous tointravenously administer the antibody of the present invention normallyat a single dose of about 0.01 to about 20 mg/kg body weight, morepreferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05to about 3 mg/kg body weight. Depending on the severity of thecondition, the frequency and the duration of the treatment can beadjusted. Effective dosages and schedules for administering Ang-2antibodies may be determined empirically; for example, patient progresscan be monitored by periodic assessment, and the dose adjustedaccordingly. Moreover, interspecies scaling of dosages can be performedusing well-known methods in the art (e.g., Mordenti et al., 1991,Pharmaceut. Res. 8:1351).

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al. 1987, J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The composition may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

A pharmaceutical composition of the present invention can be delivered,e.g., subcutaneously or intravenously with a standard needle andsyringe. In addition, with respect to subcutaneous delivery, a pendelivery device readily has applications in delivering a pharmaceuticalcomposition of the present invention. Such a pen delivery device can bereusable or disposable. A reusable pen delivery device generallyutilizes a replaceable cartridge that contains a pharmaceuticalcomposition. Once all of the pharmaceutical composition within thecartridge has been administered and the cartridge is empty, the emptycartridge can readily be discarded and replaced with a new cartridgethat contains the pharmaceutical composition. The pen delivery devicecan then be reused. In a disposable pen delivery device, there is noreplaceable cartridge. Rather, the disposable pen delivery device comesprefilled with the pharmaceutical composition held in a reservoir withinthe device. Once the reservoir is emptied of the pharmaceuticalcomposition, the entire device is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but certainlyare not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK),DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland),HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly andCo., Indianapolis, Ind.), NOVOPEN™ I, II and III (Novo Nordisk,Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen,Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPEN™,OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis,Frankfurt, Germany), to name only a few. Examples of disposable pendelivery devices having applications in subcutaneous delivery of apharmaceutical composition of the present invention include, butcertainly are not limited to the SOLOSTAR™ pen (sanofi-aventis), theFLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly).

For the treatment of eye disorders, the antibodies and antigen-bindingfragments of the invention may be administered, e.g., by eye drops,subconjunctival injection, subconjunctival implant, intravitrealinjection, intravitreal implant, sub-Tenon's injection or sub-Tenon'simplant.

The pharmaceutical composition can be also delivered in a vesicle, inparticular a liposome (see Langer 1990 Science 249:1527-1533; Treat etal. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365;Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton 1987 CRC Crit. Ref. Biomed. Eng. 14:201). Inanother embodiment, polymeric materials can be used. In yet anotherembodiment, a controlled release system can be placed in proximity ofthe composition's target, thus requiring only a fraction of the systemicdose (see, e.g., Goodson, 1984, in Medical Applications of ControlledRelease, supra, vol. 2, pp. 115-138).

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid antibodycontained is generally about 5 to about 500 mg per dosage form in a unitdose; especially in the form of injection, it is preferred that theaforesaid antibody is contained in about 5 to about 100 mg and in about10 to about 250 mg for the other dosage forms.

Therapeutic Uses of the Antibodies

The antibodies of the invention are useful, inter alia, for thetreatment, prevention and/or amelioration of any disease or disorderassociated with Ang-2 activity, including diseases or disordersassociated with angiogenesis. The antibodies and antigen-bindingfragments of the present invention may be used to treat, e.g., primaryand/or metastatic tumors arising in the brain and meninges, oropharynx,lung and bronchial tree, gastrointestinal tract, male and femalereproductive tract, muscle, bone, skin and appendages, connectivetissue, spleen, immune system, blood forming cells and bone marrow,liver and urinary tract, and special sensory organs such as the eye. Incertain embodiments, the antibodies and antigen-binding fragments of theinvention are used to treat one or more of the following cancers: renalcell carcinoma, pancreatic carcinoma, breast cancer, prostate cancer,malignant gliomas, osteosarcoma, colorectal cancer, malignantmesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer,non-small cell lung cancer, synovial sarcoma, thyroid cancer, ormelanoma.

The antibodies and antigen-binding fragments of the present inventionmay also be useful for the treatment of one or more eye disorders.Exemplary eye disorders that can be treated with the antibodies andantigen-binding fragments of the invention include, e.g., age-relatedmacular degeneration, diabetic retinopathy, and other eye disordersassociated with choroidal neovascularization, vascular leak, retinaledema and inflammation. Additionally, the anti-Ang-2 antibodies of theinvention may be administered as an adjuvant to glaucoma surgery toprevent early hem- and lymphangiogenesis and macrophage recruitment tothe filtering bleb after glaucoma surgery, and improve clinical outcome.

In other embodiments of the present invention, the antibodies orantigen-binding fragments are used to treat hypertension, diabetes(including non insulin dependent diabetes mellitus), psoriasis,arthritis (including rheumatoid arthritis), asthma, sepsis, kidneydisease and edema associated with injury, stroke or tumor.

Ang-2 expression has been shown to correlate with the severity ofvarious inflammatory and/or infectious diseases (see, e.g., Siner etal., 2009, Shock 31:348-353; Yea et al., 2008, Proc. Natl. Acad. Sci.(USA):105:17097-17102). Accordingly, the anti-Ang-2 antibodies of thepresent invention can be used to treat, prevent or ameliorate one ormore inflammatory or infectious diseases. Exemplary infectious diseasesthat can be treated, prevented or ameliorated by administration of theanti-Ang-2 antibodies of the invention include, but are not limited to:malaria (Plasmodium falciparum infection), viral hemorrhagic fevers(e.g., dengue fever), rickettsial infection, toxic shock syndrome,sepsis, hepatitis C, Bartonella bacilliformis infection, leishmaniasis,mycobacterial infection, and Epstein-Barr virus infection.

Combination Therapies

Combination therapies may include an anti-Ang-2 antibody of theinvention and, for example, another Ang-2 antagonist (e.g., ananti-Ang-2 antibody, peptibody, or CovX-body such as CVX-060 (see U.S.Pat. No. 7,521,425)). The anti-Ang-2 antibodies of the invention mayalso be administered together with another anti-angiogenic agent suchas, e.g., a VEGF antagonist (e.g., a VEGF-Trap, see, e.g., U.S. Pat. No.7,087,411 (also referred to herein as a “VEGF-inhibiting fusionprotein”), anti-VEGF antibody (e.g., bevacizumab), a small moleculekinase inhibitor of VEGF receptor (e.g., sunitinib, sorafenib orpazopanib), an anti-DLL4 antibody (e.g., an anti-DLL4 antibody disclosedin US 2009/0142354 such as REGN421), etc.), or with an antagonist ofepidermal growth factor receptor (EGFR) (e.g., anti-EGFR antibody orsmall molecule inhibitor of EGFR activity). Other agents that may bebeneficially administered in combination with the anti-Ang-2 antibodiesof the invention include cytokine inhibitors, including small-moleculecytokine inhibitors and antibodies that bind to cytokines such as IL-1,IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17,IL-18, or to their respective receptors. The present invention alsoincludes therapeutic combinations comprising any of the anti-Ang-2antibodies mentioned herein and an inhibitor of one or more of VEGF,DLL4, EGFR, or any of the aforementioned cytokines, wherein theinhibitor is an aptamer, an antisense molecule, a ribozyme, an siRNA, apeptibody, a nanobody or an antibody fragment (e.g., Fab fragment;F(ab′)₂ fragment; Fd fragment; Fv fragment; scFv; dAb fragment; or otherengineered molecules, such as diabodies, triabodies, tetrabodies,minibodies and minimal recognition units). The anti-Ang-2 antibodies ofthe invention may also be administered in combination with antivirals,antibiotics, analgesics, corticosteroids and/or NSAIDs. The anti-Ang-2antibodies of the invention may also be administered as part of atreatment regimen that also includes radiation treatment and/orconventional chemotherapy. When combined with one or more additionalagents, the anti-Ang-2 antibodies of the invention may be administeredprior to, simultaneous with (e.g., in the same formulation or inseparate formulations), or subsequent to the administration of the otheragent(s).

Diagnostic Uses of the Antibodies

The anti-Ang-2 antibodies of the present invention may also be used todetect and/or measure Ang-2 in a sample, e.g., for diagnostic purposes.For example, an anti-Ang-2 antibody, or fragment thereof, may be used todiagnose a condition or disease characterized by aberrant expression(e.g., over-expression, under-expression, lack of expression, etc.) ofAng-2. Exemplary diagnostic assays for Ang-2 may comprise, e.g.,contacting a sample, obtained from a patient, with an anti-Ang-2antibody of the invention, wherein the anti-Ang-2 antibody is labeledwith a detectable label or reporter molecule. Alternatively, anunlabeled anti-Ang-2 antibody can be used in diagnostic applications incombination with a secondary antibody which is itself detectablylabeled. The detectable label or reporter molecule can be aradioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I; a fluorescent orchemiluminescent moiety such as fluorescein isothiocyanate, orrhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase,horseradish peroxidase, or luciferase. Specific exemplary assays thatcan be used to detect or measure Ang-2 in a sample include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), andfluorescence-activated cell sorting (FACS).

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1 Generation of Human Antibodies to Human Ang-2

Human Ang-2 antigen was administered directly, with an adjuvant tostimulate the immune response, to a VELOCIMMUNE® mouse comprising DNAencoding human Immunoglobulin heavy and kappa light chain variableregions. The antibody immune response was monitored by an Ang-2-specificimmunoassay. When a desired immune response was achieved splenocyteswere harvested and fused with mouse myeloma cells to preserve theirviability and form hybridoma cell lines. The hybridoma cell lines werescreened and selected to identify cell lines that produce Ang-2-specificantibodies. Using this technique several anti-Ang-2 chimeric antibodies(i.e., antibodies possessing human variable domains and mouse constantdomains) were obtained; exemplary antibodies generated in this mannerwere designated as follows: H1M724, H1M727, H1M728, H2M730, H1M732,H1M737, H2M742, H2M743, H2M744, H1M749, H2M750 and H1M810.

Anti-Ang-2 antibodies were also isolated directly from antigen-positiveB cells without fusion to myeloma cells, as described in U.S.2007/0280945A1. Using this method, several fully human anti-Ang-2antibodies (i.e., antibodies possessing human variable domains and humanconstant domains) were obtained; exemplary antibodies generated in thismanner were designated as follows: H1H685, H1H690, H1H691, H1H693,H1H694, H1H695, H1H696, H1H704, H1H706 and H1H707.

The biological properties of the exemplary anti-Ang-2 antibodiesgenerated in accordance with the methods of this Example are describedin detail in the Examples set forth below.

Example 2 Variable Gene Utilization Analysis

To analyze the structure of antibodies produced, the nucleic acidsencoding antibody variable regions were cloned and sequenced. From thenucleic acid sequence and predicted amino acid sequence of theantibodies, gene usage was identified for each heavy chain variableregion (HCVR) and light chain variable region (LCVR) (Table 1).

TABLE 1 Antibody Identifier HCVR LCVR HCVR/ Antibody V_(H) D_(H) J_(H)V_(K) J_(K) LCVR SEQ ID NOs H1H685 3-13  3-16 4 3-20 1  2/10 H1H690 3-234-4 3 3-11 4 26/34 H1H691 3-9   4-17 6 3-20 4 50/58 H1H693 3-23 4-4 31-12 1 74/82 H1H694 3-15 6-6 4 1-5  1  98/106 H1H695 3-33  5-12 6 3-15 5122/130 H1H696 3-11  4-17 4 1-16 4 146/154 H1H704 3-33 6-6 4 1-16 5170/178 H1H706 3-33 3-3 3 1-16 1 194/202 H1H707 3-33 3-3 3 3-20 4218/226 H1M724 3-33 3-3 5 1-17 4 266/274 H1M727 1-18 3-3 6 2-28 2338/346 H1M728 3-7   6-19 4 1-5  1 290/298 H2M730 3-7   6-13 4 1-5  1362/370 H1M732 3-15 1-7 4 1-17 3 242/250 H2M742 3-23 5-5 5 2-28 4386/394 H2M743 3-23 2-8 4 1-12 4 410/418 H2M744 1-18 4-4 5 1-12 4434/442 H1M749 3-33 5-5 4 3-15 1 314/322 H2M750 3-33 6-6 4 1-16 4458/466 H1M810 3-23 3-3 3 1-12 1 482/490

Table 2 sets forth the heavy and light chain variable region amino acidsequence pairs of selected anti-Ang-2 antibodies and their correspondingantibody identifiers. The N, P and G designations refer to antibodieshaving heavy and light chains with identical CDR sequences but withsequence variations in regions that fall outside of the CDR sequences(i.e., in the framework regions). Thus, N, P and G variants of aparticular antibody have identical CDR sequences within their heavy andlight chain variable regions but contain modifications within theframework regions.

TABLE 2 HCVR/LCVR HCVR/LCVR HCVR/LCVR Name SEQ ID NOs Name SEQ ID NOsName SEQ ID NOs H1H685N  2/10 H1H685P 18/20 H1H685G 22/24 H1H690N 26/34H1H690P 42/44 H1H690G 46/48 H1H691N 50/58 H1H691P 66/68 H1H691G 70/72H1H693N 74/82 H1H693P 90/92 H1H693G 94/96 H1H694N  98/106 H1H694P114/116 H1H694G 118/120 H1H695N 122/130 H1H695P 138/140 H1H695G 142/144H1H696N 146/154 H1H696P 162/164 H1H696G 166/168 H1H704N 170/178 H1H704P186/188 H1H704G 190/192 H1H706N 194/202 H1H706P 210/212 H1H706G 214/216H1H707N 218/226 H1H707P 234/236 H1H707G 238/240 H1M724N 266/274 H1M724P282/284 H1M724G 286/288 H1M727N 338/346 H1M727P 354/356 H1M727G 358/360H1M728N 290/298 H1M728P 306/308 H1M728G 310/312 H2M730N 362/370 H2M730P378/380 H2M730G 382/384 H1M732N 242/250 H1M732P 258/260 H1M732G 262/264H1M737N 506/X*  H1M737P 514/X*  H1M737G 516/X*  H2M742N 386/394 H2M742P402/404 H2M742G 406/408 H2M743N 410/418 H2M743P 426/428 H2M743G 430/432H2M744N 434/442 H2M744P 450/452 H2M744G 454/456 H1M749N 314/322 H1M749P330/332 H1M749G 334/336 H2M750N 458/466 H2M750P 474/476 H2M750G 478/480H1M810N 482/490 H1M810P 498/500 H1M810G 502/504 *The amino acid sequenceof the LCVR of H1M737 is not shown.Control Constructs Used in the Following Examples

Various control constructs (anti-Ang-2 antibodies and anti-Ang-2peptibodies) were included in the following experiments for comparativepurposes. The control constructs are designated as follows: Control I: ahuman anti-Ang-2 antibody with heavy and light chain variable domainshaving the amino acid sequences of the corresponding domains of“Ab536(THW),” as set forth in US 2006/0018909 (see also Oliner et al.,2004, Cancer Cell 6:507-516); Control II: a peptibody that binds humanAng-2 having the amino acid sequence of “2XCon4(C),” as set forth inU.S. Pat. No. 7,205,275, (see also Oliner et al., 2004, Cancer Cell6:507-516); Control III: a peptibody that binds human Ang-2 having theamino acid sequence of “L1-7,” as set forth in U.S. Pat. No. 7,138,370;Control IV: a human anti-Ang-2 antibody with heavy and light chainvariable regions having the amino acid sequences of the correspondingdomains of “3.19.3” as set forth in US 2006/0246071; and Control V: ahuman anti-Ang-2 antibody with heavy and light chain variable regionshaving the amino acid sequences of the corresponding domains of“MEDI1/5” as set forth in WO 2009/097325. (Not all control constructswere used in every Example). In the tables that follow, the notations“Ab” and “Pb” are included to identify antibody and peptibody controls,respectively (i.e., Control I=Ab; Control II=Pb; Control III=Pb; ControlIV=Ab; and Control V=Ab).

Example 3 Antigen Binding Affinity Determination

Equilibrium dissociation constants (K_(D) values) for the binding ofselected purified Ang-2 antibodies to dimeric fibrinogen-like domain ofhuman (SEQ ID NO: 519), mouse (Mus musculus; SEQ ID NO: 520) and monkey(Macca fascicularis; SEQ ID NO: 521) Ang-2 (Ang-2FD) conjugated to humanIgG1 (SEQ ID NO:528) were determined by surface kinetics using areal-time biosensor surface plasmon resonance assay. Antibody wascaptured on a goat anti-mouse IgG polyclonal antibody surface, a goatanti-human K polyclonal antibody (Southern Biotech, Birmingham, Ala.)surface or a goat anti-human IgG polyclonal antibody (Jackson ImmunoResearch Lab, West Grove, Pa.) surface created through direct aminecoupling to a BIACORE™ CM5 sensor chip to form a captured antibodysurface. Varying concentrations (ranging from 50 nM to 6.25 nM) ofprotein were injected at 100 μl/min over captured antibody surface for90 seconds. Antigen-antibody binding and dissociation were monitored inreal time at room temperature. Kinetic analysis was performed tocalculate K_(D) and half-life of antigen/antibody complex dissociation.The results are summarized in Table 3 below.

TABLE 3 Dimeric Dimeric Dimeric Monkey Human Ang-2FD Mouse Ang-2FDAng-2FD T_(1/2) T_(1/2) T_(1/2) Antibody K_(D) (pM) (min) K_(D) (pM)(min) K_(D) (pM) (min) H1M724N 179 42.7 694 16 730 25.7 H1M728N 137 58.45650 9.9 1580 69.5 H2M730N 210 47 — — 842 36.6 H1M732N 484 35.5 170021.4 7330 24.1 H1M737N 251 34.5 1740 6.3 3810 16 H2M742N 295 38 610 30.86170 28.5 H2M743N 154 167 882 195.2 234 169.2 H2M744N 98.9 109.1 143223.1 500 281.7 H2M749N 165 42.9 529 25.5 1500 40.9 H2M750N 362 32.2 — —1470 23

The above experiment was repeated using selected purified anti-Ang-2antibodies cloned onto human IgG1. The results are summarized in Table 4below.

TABLE 4 Dimeric Dimeric Dimeric Human Ang-2FD Mouse Ang-2FD MonkeyAng-2FD Antibody K_(D) (pM) T_(1/2) (min) K_(D) (pM) T_(1/2) (min) K_(D)(pM) T_(1/2) (min) H1H685P 71.4 229.4 148 128.7 99.4 177.1 H1H690P 79126.1 91.3 105.2 55.6 195.2 H1H691P 220 38.5 220 43.8 290 41 H1H693P 50037.1 446 63.7 1170 17.6 H1H694P 126 265.6 237 166.5 356 85.6 H1H695P 245147 347 124.2 440 84.1 H1H696P 289 38.8 402 37.6 354 36.6 H1H704P 33186.1 484 61.9 818 33.5 H1H706P 201 50.4 357 47 164 53.3 H1H707P 262 26.6328 34.4 283 22.3 H1H724N 115 107 185 84 239 173 H1H728N 162 81 5760 202000 77 H1H730N 234 62 97.1 90 3400 87 H1H732N 386 57 529 51 439 118H1H742N 186 65 276 58 683 93 H1H743N 88.2 254 124 233 96.5 780 H1H744N114 127 158 115 346 164 H1H749N 118 109 177 96 407 143 H1H750N 164 127218 121 199 244 Control I (Ab) 339 34.8 339 47.1 537 27.1

Additional binding experiments were conducted using selected anti-Ang-2antibodies at two different temperatures to further assess cross-speciesaffinity. Each selected antibody or control construct was captured at aflow rate of 40 μL/min for 1 minute on a goat anti-human kappapolyclonal antibody surface created through direct chemical coupling toa BIACORE™ chip to form a captured antibody surface. Human, monkey andmouse Ang-2FD-Fc at a concentration of 25 nM or 0.78 nM was injectedover the captured antibody surface at a flowrate of 60 μL/min for 3minutes, and antigen-antibody dissociation was monitored in real timefor 20 minutes at either 25° C. or 37° C.

Results are summarized in Tables 5 (25° C. binding) and 6 (37° C.binding) below.

TABLE 5 Binding at 25° C. Dimeric Dimeric Dimeric Human Ang-2FD-mFcMouse Ang-2FD-hFc Monkey Ang-2FD-hFc Antibody K_(D) (Molar) T_(1/2)(min) K_(D) (Molar) T_(1/2) (min) K_(D) (Molar) T_(1/2) (min) H1H685P1.17E−11 227 6.51E−11 208 2.20E−11 275 H1H744N 1.16E−10 23 3.85E−10 332.44E−10 24 Control I (Ab) 1.07E−09 15 1.07E−09 15 1.03E−09 4 Control IV(Ab) 1.27E−11 269 4.02E−11 289 1.55E−11 342

TABLE 6 Binding at 37° C. Dimeric Dimeric Dimeric Human Ang-2FD-mFcMouse Ang-2FD-hFc Monkey Ang-2FD-hFc Antibody K_(D) (Molar) T_(1/2)(min) K_(D) (Molar) T_(1/2) (min) K_(D) (Molar) T_(1/2) (min) H1H685P2.70E−11 60 9.39E−11 64 7.21E−11 65 H1H744N 1.05E−10 18 2.15E−10 263.20E−10 11 Control I (Ab) — — 3.90E−10 12 — — Control IV (Ab) 9.91E−12184  5.40E−11 119 4.74E−11 107 

In another experiment, K_(D) values for selected purified antibodiesthat bind to a human “bow-Ang-2” tetrameric construct (“hBA2”) weredetermined (using the methods described above). hBA2 consists of twodimers, each dimer containing two Ang-2 fibronectin-like domainsconnected to one another by a human Fc domain. The amino acid sequenceof the dimer constituents of hBA2 is represented by SEQ ID NO:522. Theresults are summarized in Table 7 below.

TABLE 7 hBA2 Antibody K_(D) (pM) T_(1/2) (min) H1H685P 11.9 587.2H1H690P 17.9 299.3 H1H691P 106 50.6 H1H693P 299 28.7 H1H694P 68.4 111.3H1H695P 40.1 254.3 H1H696P 111 51.5 H1H704P 93.9 117.7 H1H706P 79.1 63.9H1H707P 75.2 51.4 H1H724N 23.3 323 H1H728N 41.8 185 H1H730N 55.9 152H1H732N 132 73 H1H742N 72.1 87 H1H743N 9.71 1118 H1H744N 17.2 442H1H749N 32.5 235 H1H750N 36.9 284 Control I (Ab) 83 57.5

In yet another experiment, K_(D) values for selected purified antibodiesthat bind to wild-type human Ang-2 (hAng-2-WT; SEQ ID NO: 518) and thefibrinogen-like domain of human Ang2 (hAng-2FD) were determined (asdescribed above). The results are summarized in Table 8 below.

TABLE 8 Monomeric hAng-2FD hAng-2-WT Antibody K_(D) (nM) T_(1/2) (min)K_(D) (pM) T_(1/2) (min) H1M724N 1.75 17.4 33.1 568 H1M728N 1.17 33.933.8 725 H2M730N 2.06 24.4 49.2 519 H1M732N 6.13 18.7 131 333 H1M737N2.82 13.1 59.3 282 H2M742N 4.81 18.0 67.9 437 H2M743N 0.399 156.7 14.32366 H2M744N 0.475 89.3 28.9 846 H2M749N 1.38 27.9 49 479 H2M750N 4.4221.5 40.8 991 H1H685P 0.578 55 47.6 1000 H1H691P 11 0.57 19.1 684.6H1H690P 0.594 25.16 12.4 1568 H1H693P 44.8 0.61 425 100 H1H694P 7.899.85 158 209.7 H1H695P 1.12 50.59 31.1 1770.7 H1H696P 38.4 0.20 40.3642.7 H1H704P 0.39 3.31 36.2 747.6 H1H706P 11 1.02 27.4 661.9 H1H707P145 — 77.1 217.4 H1H724N 2.4 13.34 22.6 895 H1H728N 1.18 5.86 43 566H1H730N 2.84 3.44 47.5 534 H1H732N 264 0.22 202 264 H1H742N 486 2.2944.9 666 H1H743N 2.35 33.03 9.48 3927 H1H744N 1.02 42.14 30.8 837H1H749N 1.13 33.48 12.5 1833 H1H750N 0.787 30.20 9.5 4442 Control I (Ab)44.5 0.03 47.6 512 Control II (Pb) 90 — 44.7 334.8

Additional experiments were conducted to measure the binding propertiesof selected anti-Ang-2 antibodies to monomeric hAng-2FD at 25° C. and37° C. Each selected antibody or control construct was captured at aflow rate of 40 μL/min for 1 minute on a goat anti-human IgG polyclonalantibody surface created through direct chemical coupling to a BIACORE™chip to form a captured antibody surface. Human Ang-2FD at aconcentration of 500 nM or 7.8 nM was injected over the capturedantibody surface at a flowrate of 60 μL/min for 3 minutes, andantigen-antibody dissociation was monitored in real time for 20 minutesat either 25° C. or 37° C.

Results are summarized in Tables 9 (25° C.) and 10 (37° C.) below.N/D=not determined.

TABLE 9 Binding to monomeric hAng-2FD at 25° C. ka (Ms⁻¹) kd (s⁻¹) K_(D)(Molar) T½ H1H685P 2.44E+05 7.96E−05 3.36E−10 145 minutes H1H744N2.92E+05 1.24E−04 4.24E−10  93 minutes Control I (Ab) 4.00E+05 5.10E−021.28E−07  14 seconds Control II (Pb) steady-state steady-state 9.00E−08steady-state Control III (Pb) 5.40E+05 6.30E−02 1.17E−07  11 secondsControl IV 2.84E+05 3.56E−02 1.25E−07  19 seconds (Ab)

TABLE 10 Binding to monomeric hAng-2FD at 37° C. ka (Ms⁻¹) kd (s⁻¹)K_(D) (Molar) T½ H1H685P 4.06E+05 1.39E−04 3.42E−10 83 minutes H1H744N3.86E+05 5.48E−04 1.42E−09 21 minutes Control I (Ab) steady-statesteady-state 1.51E−07 steady-state Control II (Pb) N/D N/D N/D N/DControl III (Pb) steady-state steady-state 2.94E−07 steady-state ControlIV steady-state steady-state 9.40E−08 steady-state (Ab)

As shown in this Example, several of the anti-Ang-2 antibodies generatedin accordance with the methods of Example 1 bound to Ang-2 constructswith equivalent or higher affinities than the controls. For example,antibodies H1H685, H1H690, H1H724 and H1H744 bound to dimeric humanAng-2-FD with K_(D)'s of 71.4, 79, 115, and 114 pM, respectively,whereas Control I antibody bound to dimeric human Ang-2-FD with a K_(D)of 339 pM (see Table 4). Similarly, antibodies H1H685, H1H690, H1H724and H1H744 bound to human BA2 (a tetrameric Ang-2 fibrinogen-like domainconstruct) with K_(D)'s of 11.9, 17.9, 23.3 and 17.2 pM, respectively,whereas Control I antibody bound to hBA2 with a K_(D) of 83 pM (seeTable 7). Thus, as compared to the control constructs, many of theantibodies of the invention exhibit enhanced binding to Ang-2. AntibodyH1H685P showed especially robust binding properties to Ang-2 as comparedto the control constructs.

Example 4 Preferential Binding to Ang-2 Over Ang-1

Binding experiments (plasmon resonance assays) were conducted toascertain whether selected antibodies bound to both Ang-2 and Ang-1 orif they preferentially bound to Ang-2 only. Each selected antibody orcontrol construct was captured at a flow rate of 40 μL/min for 1 minuteon a goat anti-human IgG polyclonal antibody surface created throughdirect chemical coupling to a BIACORE™ chip to form a captured antibodysurface. Full-length wild-type human Ang-1 or Ang-2 at a concentrationof 25 nM or 0.78 nM were injected over the captured antibody surface ata flowrate of 60 μL/min for 3 minutes, and antigen-antibody dissociationwas monitored in real time for 20 minutes at either 25° C. or 37° C.

The results of these experiments are summarized in Tables 11-14 below.N/D=not determined. “No binding” means that no detectable binding wasobserved under the particular experimental conditions used in theseexperiments.

TABLE 11 Binding to hAng-2-WT at 25° C. T½ ka (Ms⁻¹) kd (s⁻¹) K_(D)(Molar) (minutes) H1H685P 6.59E+05 1.60E−05 2.42E−11 722 H1H744N7.65E+05 2.57E−05 3.35E−11 450 Control I (Ab) 4.74E+05 2.26E−05 4.76E−11512 Control II (Pb) 7.73E+05 3.45E−05 4.47E−11 335 Control III (Pb)3.29E+05 1.98E−05 6.01E−11 584 Control IV (Ab) 3.80E+06 2.74E−047.22E−11 42 H1H685P 1.15E+05 8.50E−06 7.39E−11 1359 Control II (Pb)8.30E+04 5.41E−05 6.52E−10 213 Control V (Ab) 1.12E+05 2.66E−05 2.73E−10434

TABLE 12 Binding to hAng-1-WT at 25° C. T½ ka (Ms⁻¹) kd (s⁻¹) K_(D)(Molar) (minutes) H1H685P No binding No binding No binding No bindingH1H744N 4.10E+05 3.81E−05 9.30E−11 303 Control I (Ab) 4.55E+05 2.49E−055.47E−11 464 Control II (Pb) 4.53E+05 3.54E−05 7.82E−11 326 Control III(Pb) No binding No binding No binding No binding Control IV 6.60E+051.11E−04 1.68E−10 105 (Ab) H1H685P No binding No binding No binding Nobinding Control II (Pb) 3.04E+05 2.51E−05 8.26E−11 460 Control V (Ab)2.75E+05 6.68E−05 2.43E−10 173

TABLE 13 Binding to hAng-2-WT at 37° C. ka (Ms⁻¹) kd (s⁻¹) K_(D) (Molar)T½ (minutes) H1H685P 8.54E+05 3.76E−05 4.40E−11 707 H1H744N 7.01E+052.43E−04 3.47E−10 48 H1H685P 1.36E+05 2.16E−05 1.59E−10 535 Control II(Pb) 3.79E+04 1.17E−04 3.09E−09 99 Control V (Ab) 9.42E+04 7.92E−058.41E−10 146

TABLE 14 Binding to hAng-1-WT at 37° C. T½ ka (Ms⁻¹) kd (s⁻¹) K_(D)(Molar) (minutes) H1H685P No binding No binding No binding No bindingH1H744N 1.47E+06 5.20E−05 3.12E−11 222 Control III (Pb) No binding Nobinding No binding No binding H1H685P No binding No binding No bindingNo binding Control II (Pb) 2.81E+05 4.35E−05 1.55E−10 266 Control V (Ab)4.42E+05 5.47E−05 1.24E−10 211

These results show that H1H685P is unique among the antibodies tested inthis experiment in that it binds with high affinity to Ang-2 but doesnot bind to Ang-1. The only other construct that exhibits binding toAng-2 but not to Ang-1 is Control III. It should be emphasized, however,that Control III is a peptibody and that all of the other antibodiestested in this experiment bound to both Ang-2 and Ang-1. The selectivityfor Ang-2 binding may confer therapeutic benefits on H1H685P that arenot possessed by antibodies that bind to both Ang-2 and Ang-1.

Example 5 Inhibition of Ang-2 Binding to Human Tie-2

Tie-2 is a natural receptor for Ang-2. Anti-Ang-2 antibodies were testedfor their ability to block Ang-2 binding to human Tie-2 (hTie-2).hTie-2-mFc (a chimeric construct consisting of human Tie-2 conjugated tomouse IgG; SEQ ID NO:525) was coated onto 96-well plates at aconcentration of 2 μg/ml and incubated overnight followed by washingfour times in wash buffer (PBS with 0.05% Tween-20). The plate was thenblocked with PBS (Irvine Scientific, Santa Ana, Calif.) containing 0.5%BSA (Sigma-Aldrich Corp., St. Louis, Mo.) for one hour at roomtemperature. In a separate plate, purified anti-Ang-2 antibodies, at astarting concentration of 50 nM, were serially diluted by a factor ofthree across the plate. Human, mouse or monkey Ang-2FD proteinconjugated to human IgG (Ang-2FD-hFc) were added to final concentrationsof 2 nM, 8 nM, or 2 nM respectively and incubated for one hour at roomtemperature. The antibody/Ang-2FD-Fc mixture was then added to the platecontaining hTie-2-mFc and incubated for one hour at room temperature.Detection of Ang-2FD-hFc bound to hTie-2-mFc protein was determined withHorse-Radish Peroxidase (HRP) conjugated to a-human IgG antibody(Jackson Immuno Research Lab, West Grove, Pa.) and developed by standardcolorimetric response using tetramethylbenzidine (TMB) substrate (BDBiosciences, San Jose, Calif.). Absorbance was read at OD₄₅₀ for 0.1sec. Percent blocking of Ang-2FD-hFc binding to hTie-2-mFc by 16.67 nMof selected anti-Ang-2 antibodies is shown in Table 15.

TABLE 15 Percent Blocking of Ang-2FD Binding to Tie-2 Human Mouse MonkeyAntibody Ang-2FD-hFc Ang-2FD-hFc Ang-2FD-hFc H1M724N 99.5 96.6 95.2H1M728N 98.5 83.9 97.1 H2M730N 98.9 55.0 97.3 H1M732N 97.7 90.9 95.8H1M737N 99.1 95.4 90.5 H2M742N 99.6 98.6 94.1 H2M743N 99.6 98.4 95.1H2M744N 99.5 98.4 95.5 H2M749N 99.5 97.3 97.4 H2M750N 99.4 53.7 97.4Control I (Ab) 94.5 90.2 96.9

In a similar experiment, selected purified anti-Ang-2 antibodies clonedonto human IgG1 were tested for their ability to block Ang-2FD bindingto hTie-2 (as described above). Percent blocking of Ang-2FD-hFc bindingto hTie-2-mFc by 16.67 nM of selected anti-Ang-2 antibodies is shown inTable 16. NT: not tested.

TABLE 16 Percent Blocking of Ang-2FD Binding to Tie-2 Human Mouse MonkeyAntibody Ang-2FD-hFc Ang-2FD-hFc Ang-2FD-hFc H1H685P 93.8 97.1 62.2H1H690P 97.2 98.0 99.6 H1H691P 97.4 96.7 99.8 H1H693P 73.9 63.6 NTH1H694P 79.8 36.0 NT H1H695P 98.4 97.6 NT H1H696P 98.2 94.9 99.2 H1H704P97.0 41.8 NT H1H706P 97.1 95.9 99.8 H1H707P 95.1 93.8 NT H1H724N 96.697.1 96.6 H1H744N 97.9 97.6 96.2 Control I (Ab) 97.3 82.2 98.5

In another experiment, selected purified anti-Ang-2 antibodies weretested for their ability to block binding of 20 pM biotinylated hBA2 tohTie-2 (as described above). For this experiment, human Tie-2 conjugatedto a histidine tag (hTie-2-His; SEQ ID NO:526) was used in a similarfashion to the hTie-2-mFc described above. Antibody concentrations from5 nM were serially diluted three-fold. An IC₅₀ (InhibitoryConcentration) value was generated by calculating the amount of antibodyrequired to block 50% of the signal from the binding of biotin-hBA2 toTie-2. An average IC₅₀ value for each antibody was calculated based ontwo separate experiments. The results are summarized in Table 17. NB: noblocking observed at 5 nM concentration.

TABLE 17 Biotin-hBA2 Antibody Average IC₅₀ (pM) H1M724N 9.72 H1M728N14.05 H2M730N 14.60 H1M732N 82.17 H1M737N 13.01 H2M742N 9.65 H2M743N11.01 H2M744N 11.43 H2M749N 6.43 H2M750N 8.83 Control I (Ab) 30.23Control II (Pb) 7.75 Control III (Pb) 16.49

In a similar experiment, selected purified anti-Ang-2 antibodies clonedonto human IgG1 were tested for their ability to block binding ofbiotinylated hBA2 to hTie-2 (as described above). The results are shownin Table 18. NB: no blocking observed at 5 nM concentration.

TABLE 18 Biotin-hBA2 Antibody IC₅₀ (pM) H1H685P 20 H1H690P 17 H1H691P 13H1H693P NB H1H694P NB H1H695P 59 H1H696P 22 H1H704P 56 H1H706P 8 H1H707P22 H1H724N 4 H1H744N 25

This Example illustrates that several of the anti-Ang-2 antibodiesgenerated in accordance with the methods of Example 1 blocked theinteraction between the Ang-2 fibrinogen-like domain and its receptor(TIE-2) to an equivalent or greater extent than the control antibody.For example, antibodies H1H690, H1H691, H1H695, H1H696, H1H704, H1H706,H1H707, H1H724 and H1H744 each caused greater than 95% blocking ofhuman, mouse and monkey Ang-2FD constructs to the TIE-2 receptor,similar to the results observed with the control constructs (see Table16).

Example 6 Inhibition of Full-Length Ang-2 and Ang-1 Binding to HumanTie-2

Tie-2 is a receptor for Ang-1 as well as Ang-2. Therefore, in thepresent Example, the ability of certain anti-Ang-2 antibodies to blockbinding of Ang-2 or Ang-1 to human Tie-2 was measured and compared.

The ELISA experiments shown in this Example were conducted in a similarmanner to the experiments of Example 5. Briefly, hTie-2-mFc (a chimericconstruct consisting of human Tie-2 conjugated to mouse IgG; SEQ IDNO:525) was coated onto 96-well plates at a concentration of 2 μg/ml andincubated overnight followed by washing four times in wash buffer (PBSwith 0.05% Tween-20). The plate was then blocked with PBS (IrvineScientific, Santa Ana, Calif.) containing 0.5% BSA (Sigma-Aldrich Corp.,St. Louis, Mo.) for one hour at room temperature. In a separate plate,purified anti-Ang-2 antibodies and control constructs, at a startingconcentration of 300 nM, were serially diluted by a factor of threeacross the plate. Full-length human Ang-2 or Ang-1 protein conjugated to6× histidine tag (R&D Systems, Minneapolis, Minn.) were added to a finalconcentration of 0.6 nM and incubated for one hour at room temperature.The antibody/antigen mixture was then added to the plate containinghTie-2-mFc and incubated for one hour at room temperature. Detection ofAng-2-His or Ang-1-His bound to hTie-2-mFc protein was determined withHorse-Radish Peroxidase (HRP) conjugated to a-Penta-His antibody(Qiagen, Valencia, Calif.) and developed by standard colorimetricresponse using tetramethylbenzidine (TMB) substrate (BD Biosciences, SanJose, Calif.). Absorbance was read at OD₄₅₀ for 0.1 sec. An IC₅₀(Inhibitory Concentration) value was generated by calculating the amountof antibody required to block 50% of the signal from the binding ofhuman Ang-2 or Ang-1 to Tie-2. The results, expressed in terms of IC₅₀are shown in Table 19, columns (1) and (2). The extent to which theantibodies or control constructs block the hAng-2/Tie-2 interactionrelative to the hAng-1/Tie-2 interaction is reflected in the folddifference in IC₅₀ shown in column (3); that is, a higher number incolumn (3) indicates a greater capacity to block the hAng-2/Tie-2interaction than the hAng-1/Tie-2 interaction.

TABLE 19 (3) Fold Difference in (1) (2) hAng-1 Blocking BlockingBlocking IC₅₀ hAng-2 hAng-1 Compared WT to WT to to h-Ang-2 AntibodyTie-2 IC₅₀ (M) Tie-2 IC₅₀ (M) Blocking IC₅₀* H1H685P1.294E−10 >3.000E−07  >2318 H1H744N 7.871E−11 1.872E−07 2378 Control I(Ab) 9.372E−11 6.171E−08 658 Control II (Pb) 3.096E−11 5.509E−11 1.8Control III (Pb) 1.626E−10 >1.000E−06  >6150 Control IV (Ab) 1.476E−104.252E−09 28.8 *Calculated by dividing the hAng-1 blocking IC₅₀ (column2) by the hAng-2 blocking IC₅₀ (column 1).

In an effort to further assess the ability of selected anti-hAng-2antibodies to block the binding of Ang-1 to Tie-2, a biosensor surfaceplasmon resonance experiment was conducted. In this experiment, a humanTie-2 full-length extracellular domain construct (hTie-2-mFc-ecto) wasamine-coupled on a BIACORE™ chip to create a receptor coated surface.Selected anti-hAng-2 antibodies and control constructs, at 1 μM(100-fold excess over antigen), were premixed with 10 nM of hAng-1-WT,followed by 60 minutes incubation at 25° C. to allow antibody-antigenbinding to reach equilibrium to form equilibrated solutions. Theequilibrated solutions were injected over the receptor surfaces at 5μL/min for 5 minutes at 25° C. Changes in resonance units (RU) due tothe binding of the hAng-1-WT to hTie-2-mFc were determined. Anirrelevant peptibody construct with no binding to hAng-1 was included inthis experiment to establish the 0% blocking baseline, and a humanTie-2-mFc construct was used as a positive control for blocking. Theamount of Ang-1 bound to Tie-2 following antibody preincubation,expressed as a percentage of the amount of Ang-1 bound to Tie-2following negative control preincubation, is shown in Table 20. (Agreater amount of Ang-1 binding to Tie-2 signifies a lower degree ofantibody blocking).

TABLE 20 Percent of Negative Control Antibody RU (average) BindingNegative Control 169 100 (irrelevant peptibody) hTie-2-mFc 71 42 H1H685P137 81 H1H744N 57 34 H1H691P 117 69 H1H706P 140 83 H1H724N 57 34 ControlI (Ab) 48 28 Control II (Pb) 48 28 Control III (Pb) 160 95

The foregoing experiment was repeated using different amounts of Ang-2blockers and controls. In particular, a human Tie-2 full-lengthextracellular domain construct (hTie-2-mFc-ecto) was amine-coupled on aBIACORE™ chip to create a receptor-coated surface. Selected anti-hAng-2antibodies and control constructs (50 or 150 nM) were mixed withhAng-2-WT (25 nM) followed by 60 minutes incubation at 25° C. to allowantibody-antigen binding to reach equilibrium. The equilibratedsolutions were injected over the receptor surfaces at 10 μL/min for 5minutes at 25° C. To evaluate the ability of the selected anti-hAng-2antibodies to block Ang-1-WT binding to hTie-2, a similar procedure wasfollowed except the antibodies were tested at three concentrations (50,100 or 1000 nM) and incubated with 10 nM of hAng-1-WT. Changes inresonance units (RU) due to the binding of the Ang-2-WT or hAng-1-WT tohTie-2-mFc were determined. An irrelevant antibody with no binding toeither angiopoietin was included in these experiments to establish the0% blocking baseline, and a human Tie-2-mFc construct was used as apositive control for blocking. Results are summarized in Tables 21(hAng-1 applied to a hTie-2 surface) and 22 (hAng-2 applied to a hTie-2surface).

TABLE 21 (hAng-1 WT) Amount of Antibody or Control 50 nM 100 nM 1000 nMPercent of Percent of Percent of Specific Neg. Ctrl Specific Neg. CtrlSpecific Neg. Ctrl Antibody Bound RU Binding Bound RU Binding Bound RUBinding Negative Control 316 100 307 100 276 100 (irrelevant antibody)hTie-2-mFc 70 22 39 13 −47 0 H1H685P 299 95 291 95 289 105 Control II(Pb) 8 2.5 4 1.3 −1 0 Control V (Ab) 150 48 114 37 29 11

TABLE 22 (hAng-2 WT) Amount of Antibody or Control 50 nM 150 nM Percentof Percent of Specific Neg. Ctrl Specific Neg. Ctrl Antibody Bound RUBinding Bound RU Binding Negative Control 281 100 278 100 (irrelevantantibody) hTie-2-mFc 97 35 82 30 H1H685P 12 4.3 12 4.3 Control II (Pb)10 3.6 10 3.6 Control V (Ab) 12 4.3 12 4.3

The results obtained from these experiments are in agreement withprevious results which showed that H1H685P preferentially binds to Ang-2over Ang-1 (see Example 4). In particular, the results from this Exampleshow that several anti-Ang-2 antibodies (e.g., H1H685P and H1H706P) donot significantly block the binding of human Ang-1 to human Tie-2, eventhough, in other experiments, it was demonstrated that these antibodiespotently blocked the interaction between Ang-2 and Tie-2 (see Example 5,Table 16). Moreover, in these experiments none of the controlconstructs, except for the Control III peptibody, exhibited the samedegree of preferential binding/blocking of Ang-2 over Ang-1 as theexemplary anti-Ang-2 antibodies of the present invention, such asH1H685P.

Example 7 Inhibition of Ang-2-Mediated Tie-2 Phosphorylation byAnti-Ang-2 Antibodies

The inventors of the present invention have demonstrated that Ang-2expression can be induced in human umbilical vein endothelial cells(HUVECs) by the transcription factor FOXO1 (Daly et al. 2006 PNAS103:15491). Further, the inventors have shown that infection of HUVECswith an adenovirus encoding FOXO1 results in expression and secretion ofAng-2, followed by activation of Tie-2 phosphorylation (Daly et al. 2006PNAS 103:15491).

Anti-Ang-2 antibodies were tested for their ability to inhibit Tie-2phosphorylation. Briefly, 7×10⁵ HUVECs (Vec Technologies, Rensselaer,N.Y.) were plated in 6 cm cell culture dishes in 3.5 ml of MCDB131Complete medium (Vec Technologies, Rensselaer, N.Y.). The following day,the cells were washed with Opti-MEM (Invitrogen Corp., Carlsbad, Calif.)and fed with 2 ml of Opti-MEM. Recombinant adenoviruses encoding eithergreen fluorescent protein (GFP; control) or human FOXO1 (Daly et al.2004 Genes Dev. 18:1060) were added to the cells at a concentration of10 pfu/cell and incubated for four hours. Cells were then washed withMCDB131 and fed with 2 ml of MCDB131 containing anti-Ang-2 antibodies ata concentration of 0.5 μg/ml. At twenty hours post infection, cells werelysed and subjected to Tie-2 immunoprecipitation as described by Daly etal., Proc. Natl. Acad. Sci. USA 103:15491-15496 (2006). Immunoglobulinwas collected on protein NG beads (Santa Cruz Biotechnology, Santa Cruz,Calif.) for one hour. Beads were washed with cold lysis buffer andresuspended in SDS sample buffer for analysis by western blot withantibodies specific for phosphotyrosine (Millipore, Billerica, Mass.) orTie-2. Signals were detected using HRP-conjugated secondary antibodiesand ECL reagents (GE Healthcare, Piscataway, N.J.). X-Ray films werescanned and the phospho-Tie-2 and Tie-2 signals were quantified usingImageJ software. The phospho-Tie-2/Tie-2 ratios were used to determinethe % inhibition for each anti-Ang-2 antibody (i.e. Percentinhibition=Reduction in phospho-Tie-2/Tie-2 as compared to control). Forexample, a reduction in Tie-2 phosphorylation to the level observed inthe control sample is considered to be 100% inhibition. Relativeinhibition (+, ++, +++) for each anti-Ang-2 antibody tested according tothe percent inhibition observed (25-50%, 50-75%, 75-100%, respectively)is shown in Table 23.

TABLE 23 Inhibition of Antibody Tie-2 phosphorylation H1H685P +++H1H690P +++ H1H691P +++ H1H693P +++ H1H694P ++ H1H695P +++ H1H696P +++H1H704P +++ H1H706P +++ H1H707P +++ H1M724N +++ H1M728N ++ H1M732N ++H1M742N ++ H1M743N +++ H1M744N +++ H1M749N ++ H1M750N +++ Control I(Ab) + Control II (Pb) +++

As demonstrated in this Example, the anti-Ang-2 antibodies generated inaccordance with the methods of Example 1 inhibited Tie-2 phosphorylationto a greater extent than the Control I antibody. Especially robustinhibition was observed with antibodies H1H685, H1H690, H1H691, H1H693,H1H695, H1H696, H1H704, H1H706, H1H707, H1M724, H1M744 and H1M750.

Example 8 Inhibition of Ang-1-Mediated Tie-2 Phosphorylation

As shown in the previous Example, Ang-2 can mediate the phosphorylationof Tie-2. Ang-1 is also capable of promoting Tie-2 phosphorylation. Inthe present Example, the ability of selected anti-Ang-2 antibodies toblock Ang-1-mediated phosphorylation of Tie-2 was assessed.

EA.hy926 cells (Edgell et al., Proc. Natl. Acad. Sci. USA 80:3734-3737(1983)) were plated at 5×10⁶ cells per 10 cm dish in 10 ml DMEM with 10%FBS, HAT, L-glutamine and penicillin/streptomycin. After 24 hours, cellswere serum-starved for 1 hour in 10 ml DMEM+1 mg/ml BSA. Cells were thenstimulated for 10 minutes with 500 ng/ml of recombinant human Ang-1 (R&DSystems) in the presence of either an irrelevant isotype controlantibody (“9E10”) at 400 nM or the anti-Ang-2 antibody H1H685P, orcontrol agents (Control I, Control II, Control IV, or Control V) atconcentrations ranging from 10 to 400 nM.

Following incubation, cells were lysed and Tie-2 was immunoprecipitatedas described by Daly et al., Proc. Natl. Acad. Sci. USA 103:15491-15496(2006). Immune complexes were collected by incubation with protein A/Gbeads (Santa Cruz Biotechnology, Santa Cruz, Calif.) for 60 min. Beadswere washed with cold lysis buffer and bound proteins were eluted byheating in SDS sample buffer. Samples were then subjected to Westernblot analysis with monoclonal antibodies against Tie-2 orphosphotyrosine (clone 4G10, Millipore, Billerica, Mass.). Results areshown in FIG. 2.

Signals were detected using HRP-conjugated secondary antibodies and ECLreagents (GE Healthcare, Piscataway, N.J.). X-ray films were scanned andthe phospho-Tie-2 and Tie-2 signals were quantified using ImageJsoftware. The phospho-Tie-2/Tie-2 ratios were used to determine the %inhibition for each antibody or peptibody. Percent inhibition=reductionin phospho-Tie-2/Tie-2 as compared to the control sample (400 nM isotypecontrol antibody).

In the presence of the control antibody 9E10, Ang-1 strongly activatedTie-2 phosphorylation (FIG. 2, panel A—compare lanes 2 and 3 vs lane 1).All of the control agents that were tested significantly inhibited Tie-2phosphorylation, with complete inhibition occurring at 50 nM for ControlII (FIG. 2, panel B—lane 17), 100 nM for Control IV (FIG. 2, panelA—lane 11) and 200 nM for Control I (FIG. 2, panel B—lane 24) andControl V (FIG. 2, panel C—lane 9). By contrast, H1H685P had nosignificant inhibitory effect even at 400 nM (FIG. 2, panel A—lanes4-8), These results provide additional confirmation of the specificityof H1H685P for Ang-2 over Ang-1.

Example 9 Inhibition of Tumor Growth by Anti-Ang-2 Antibodies

The effect of selected purified anti-Ang-2 antibodies on tumor growthwas determined using two tumor cell lines.

PC3 (Human prostate cancer cell line) Briefly, 5×10⁶ PC3 cells in 100 μlof growth factor-reduced Matrigel (BD Biosciences) were injectedsubcutaneously into the flanks of 6-8 week old male NCr nude mice(Taconic, Hudson, N.Y.). After tumor volumes reached an average of about200 mm³, mice were randomized into groups for treatment. Mice in eachtreatment group were administered an anti-Ang-2 antibody, Fc protein, orcontrol construct, at a concentration of 10 mg/kg via intraperitonealinjection twice per week for approximately three weeks (Table 24) or atconcentrations of 2.5, 12.5, or 25 mg/kg via subcuataneous injectiontwice per week for approximately three weeks (Table 25). Tumor volumeswere measured twice per week over the course of the experiment and tumorweights were measured upon excision of tumors at the conclusion of theexperiment. Averages (mean+/−standard deviation) of tumor weight andgrowth were calculated for each treatment group. Percent decrease oftumor weight and growth were calculated from comparison to Fc proteinmeasurements. Results are summarized in Tables 24 and 25.

TABLE 24 % Avg Tumor Decrease Avg Tumor % Decrease in Growth in TumorAntibody Weight (g) Tumor Weight (mm³) Growth Fc protein 0.66 ± 0.26 —509 ± 213 — Control I (Ab) 0.47 ± 0.23 29 300 ± 242 41 H1H724N 0.55 ±0.07 17 392 ± 169 23 H1H744N 0.43 ± 0.20 35 259 ± 212 49 H1H685P 0.44 ±0.12 33 305 ± 143 40 H1H691P 0.59 ± 0.07 11 485 ± 141 5 H1H706P 0.52 ±0.14 21 329 ± 125 35

TABLE 25 Avg Tumor % Decrease in Antibody Growth (mm³) Tumor Growth Fcprotein 1031 ± 485  — Control II (Pb) 356 ± 196 65 (2.5 mg/kg) ControlII (Pb) 360 ± 162 65 (12.5 mg/kg) Control II (Pb) 527 ± 218 49 (25mg/kg) H1H685P 308 ± 274 70 (2.5 mg/kg) H1H685P 550 ± 150 47 (12.5mg/kg) H1H685P 413 ± 208 60 (25 mg/kg)

As shown above, antibodies H1H744N and H1H685P demonstrated especiallymarked anti-tumor activity in the PC3 mouse tumor model as compared tothe control constructs.

The results of similar experiments using the PC3 mouse tumor model anddifferent experimental antibodies (dosed at 2 mg/kg, twice per week) areshown in Tables 26 and 27.

TABLE 26 % Decrease Avg Tumor % Decrease in Avg Tumor in Tumor AntibodyWeight (g) Tumor Weight Growth (mm³) Growth Fc protein 0.626 ± 0.156 —356 ± 93  — Control I 0.347 ± 0.093 45 250 ± 145 30 (Ab) H2M742N 0.407 ±0.076 35 220 ± 102 38 H2M743N 0.372 ± 0.122 41 179 ± 169 50

TABLE 27 % Decrease Avg Tumor % Decrease in Avg Tumor in Tumor AntibodyWeight (g) Tumor Weight Growth (mm³) Growth Fc protein 0.552 ± 0.211 —473 ± 202 — H1M749N 0.383 ± 0.275 31 220 ± 261 54 H1M750N 0.348 ± 0.12837 227 ± 195 52

COLO 205 (Human colorectal adenocarcinoma cell line) Briefly, 2×10⁶COLO205 cells in 100 μl of serum-free medium were injected subcutaneouslyinto the flank of 6-8 week old male NCr nude mice (Taconic, Hudson,N.Y.). After tumor volumes reached an average of about 150 mm³, micewere randomized into groups for treatment with antibody or Fc protein.Mice in each treatment group were administered an anti-Ang-2 antibody orFc protein at a concentration of 4 mg/kg via intraperitoneal injectiontwice per week for approximately two weeks. Tumor volumes were measuredtwice per week over the course of the experiment and tumor weights weremeasured upon excision of tumors at the conclusion of the experiment.Averages (mean+/−standard deviation) of tumor weight and growth werecalculated for each treatment group. “Avg. Tumor Growth” represents theaverage growth from the time of treatment initiation (when tumors wereapproximately 150 mm³). Percent decrease of tumor weight and growth arecalculated from comparison to Fc protein measurements. Results aresummarized in Table 28.

TABLE 28 % % Decrease Decrease Avg Tumor in Tumor Avg Tumor in TumorAntibody Weight (g) Weight Growth (mm³) Growth Fc protein 0.847 ± 0.180— 731 ± 249 — Control I (Ab) 0.503 ± 0.090 41 367 ± 121 50 Control II(Pb) 0.608 ± 0.085 28 492 ± 82  33 H1M724N 0.531 ± 0.103 37 336 ± 125 54H2M742N 0.576 ± 0.057 32 427 ± 92  42 H2M744N 0.491 ± 0.051 42 409 ± 16244 H1M749N 0.603 ± 0.142 29 449 ± 169 39

A similar experiment was carried out to assess the effect of H1H685P, inparticular, on COLO 205 tumor growth. Briefly, 2×10⁶ COLO 205 cells in100 μl of serum-free medium were implanted subcutaneously into the righthind flank of 9-11 week-old male SCID CB17 mice. When the tumors reached˜125 mm³, mice were randomized into 5 groups (n=7-8 mice/group) andtreated twice per week with Fc protein (15 mg/kg), H1H685P (5 or 25mg/kg) or Control II (5 or 25 mg/kg) for a period of 19 days. Tumorvolumes were measured twice per week over the course of the experimentand tumor weights were measured upon excision of tumors at the end ofthe experiment. Averages of tumor weight and growth from the beginningof treatment were calculated for each group. Percent decrease of tumorweight and growth are calculated from comparison to the Fc controlgroup. The results are shown in Table 29.

TABLE 29 Antibody Avg Tumor % Decrease in Avg Tumor % Decrease inAntibody Concentration Weight (g) Tumor Weight Growth (mm³) Tumor GrowthFc protein 25 mg/kg 0.800 ± 0.108 — 675 ± 93  — Control II (Pb)  5 mg/kg0.481 ± 0.091 40 288 ± 85  57 Control II (Pb) 25 mg/kg 0.393 ± 0.136 51267 ± 155 60 H1H685P  5 mg/kg 0.458 ± 0.125 43 370 ± 114 45 H1H685P 25mg/kg 0.430 ± 0.139 46 295 ± 160 56

As with the PC3 mouse tumor model, several of the antibodies of theinvention, including H1H685P, exhibited substantial anti-tumoractivities in the COLO 205 mouse model that were at least equivalent tothe anti-tumor activities exhibited by the control molecules.

Example 10 Inhibition of Tumor Growth and Perfusion by A Combination ofan Anti-Ang-2 Antibody and a VEGF Inhibitor

To determine the effect of combining an anti-Ang-2 antibody with a VEGFinhibitor on the growth of COLO 205 xenografts, 2×10⁶ cells wereimplanted subcutaneously into the right hind flank of 6-8 week-oldfemale SCID mice. When the tumors reached an average volume of −350 mm³,mice were randomized into 4 groups (n=6 mice/group) and treated with:human Fc protein (7.5 mg/kg), H1H685P (5 mg/kg), VEGF Trap (see U.S.Pat. No. 7,087,411) (2.5 mg/kg) or the combination of H1H685P+VEGF Trap.Mice were given a total of 3 doses over 10 days of treatment. Tumorvolumes were measured twice per week over the course of the experiment.Averages of tumor growth from the start of treatment (mean+/−standarddeviation) were calculated for each treatment group. Percent decrease oftumor growth was calculated from comparison to the Fc control group. Theresults are shown in Table 30. Note that in the VEGF Trap and in theH1H685P+VEGF Trap groups the average tumor size was smaller at the endof treatment than at the beginning, i.e., tumor regression was observed.

TABLE 30 Avg Tumor % Decrease in Antibody Growth (mm³) Tumor Growth Fcprotein 366 ± 65  — H1H685P 74 ± 77 80 VEGF Trap −62 ± 44  117 H1H685P +VEGF Trap −221 ± 131  160

The results of this experiment demonstrate that the combination ofH1H685P+VEGF Trap causes a decrease in tumor growth that is greater thanthe percent decrease in tumor growth caused by either component alone.

To provide additional evidence of combination efficacy, the effect ofthe H1H685P+VEGF Trap combination on the growth of MMT tumors wasassessed. 0.5×10⁶ MMT cells were implanted subcutaneously into the righthind flank of 6-8 week-old female SCID mice. When the tumors reached anaverage volume of ˜400 mm³, mice were randomized into 4 groups (n=11mice/group) and treated with: human Fc protein (17.5 mg/kg), H1H685P(12.5 mg/kg), VEGF Trap (5 mg/kg) or the combination of H1H685P+VEGFTrap. The Fc and H1H685P groups were given 3 doses over 9 days. The VEGFTrap and combination groups were given 4 doses over 12 days. Tumorvolumes were measured twice per week over the course of the experimentand tumor weights were measured upon excision of tumors at the end ofthe experiment (due to their large size, tumors from the Fc and H1H685Pgroups were collected 3 days before tumors from the VEGF Trap andcombination groups). Averages (mean+/−standard deviation) of tumorgrowth from the beginning of treatment and of tumor weight werecalculated for each group. Percent decrease of tumor weight and growthare calculated from comparison to the Fc control group. The results areshown in Table 31.

TABLE 31 % Avg Tumor Decrease Avg Tumor % Decrease in Growth in TumorAntibody Weight (g) Tumor Weight (mm³) Growth Fc protein 1.591 ± 0.265 —1337 ± 273 — H1H685P 1.409 ± 0.314 11 1135 ± 306 15 VEGF Trap 0.889 ±0.141 44  536 ± 179 60 H1H685P + 0.599 ± 0.066 62 215 ± 92 84 VEGF Trap

These results confirm the enhanced tumor inhibiting effect ofH1H685P+VEGF Trap relative to the single agent treatments.

To determine whether the combination of H1H685P+VEGF Trap has a greatereffect on tumor vessel function than the single agents, amicro-ultrasound (VisualSonics' Vevo 770 imaging system) was used toassess changes in tumor perfusion. COLO 205 tumors were grown to ˜125mm³ and mice were then treated for 24 hrs with H1H685P, VEGF Trap or thecombination of both agents. Following treatment, tumor vessel perfusionwas determined based on contrast-enhanced micro-ultrasound 2D imageacquisition and analysis of a “wash-in” curve, which represents theamount of contrast agent entering the tumor. Average (mean+/−standarddeviation) tumor perfusion was calculated for each group. Percentdecrease was calculated from comparison to the Fc control group. Theresults are shown in Table 32.

TABLE 32 Relative Tumor % Decrease in Antibody Perfusion Tumor PerfusionFc protein 8.09 ± 2.16 — H1H685P 6.32 ± 2.81 22 VEGF Trap 6.99 ± 1.36 14H1H685P + VEGF Trap 2.46 ± 0.34 70

Consistent with the enhanced effect of the combination treatment onperfusion, anti-CD31 staining of tumor sections demonstrated a morepotent effect of the combination on tumor blood vessel density (data notshown). The increased effect of the H1H685P+VEGF Trap combination on thefunction of the tumor vasculature provides a potential explanation forthe enhanced effects of the combination therapy on tumor growth.

Example 11 Inhibition of Tumor Growth by A Combination of an Anti-Ang-2Antibody and a Chemotherapeutic Agent

To test the effect of H1H685P in combination with a chemotherapeuticagent on tumor growth, 2.5×10⁶ COLO 205 tumor cells were implantedsubcutaneously into the right hind flank of 8-9 week-old male SCID mice.When the tumors reached an average volume of ˜150 mm³ (day 17 afterimplantation), mice were randomized into 4 groups (n=5 mice/group) andtreated as follows: the first group was treated sc with 15 mg/kg hFc andintraperitoneally (ip) with 5-FU vehicle; the second group was treatedsc with 15 mg/kg of H1H685P; the third group was treated ip with 75mg/kg of 5-FU; the fourth group was treated with the combination of 15mg/kg H1H685P sc plus 75 mg/kg 5-FU ip. Mice received a total of threetreatments, administered every 3-4 days. Tumor volumes were measuredtwice per week over the course of the experiment. Average(mean+/−standard deviation) tumor growth from the beginning of treatmentuntil day 38 was calculated for each group. Percent decrease of tumorgrowth was calculated from comparison to the control group. The resultsare shown in Table 33.

TABLE 33 Avg Tumor Growth % Decrease in Treatment (mm³) Tumor Growth Fcprotein + 5-FU  574 ± 110 — vehicle H1H685P 405 ± 80 29 5-FU 313 ± 60 45H1H685P + 5-FU 175 ± 78 70

The results of this experiment show that the combination of H1H685P and5-FU caused a greater decrease in tumor growth than either agentadministered separately.

Example 12 Anti-Ang-2 Antibodies Attenuate Ocular Angiogenesis In Vivo

In this Example, the effects of selected anti-Ang-2 antibodies onretinal vascularization in a mouse model was assessed.

In one set of experiments wild-type mice were used. In another set, miceexpressing a human Ang-2 in place of the wild-type mouse Ang-2(designated “hu-Ang-2 mice”) were used. The mice at two days of age (P2)were injected subcutaneously with either control Fc or with selectedanti-Ang-2 antibodies at a dose of 12.5 mg/kg. Three days later (at P5),pups were euthanized, and eyeballs were enucleated and fixed in 4% PFAfor 30 minutes. Retinas were dissected, stained with Griffoniasimplicifolia lectin-1 for 3 hours or overnight at 4° C. to visualizethe vasculature, and flat-mounted on microscope slides. Images weretaken using a Nikon Eclipse 80i microscope camera and analyzed usingAdobe Photoshop CS3, Fovea 4.0, and Scion 1.63 software.

Areas of the retina covered with superficial vasculature were measuredand used as a readout of antibody activity. The reduction in the size ofthe vascular areas in mice treated with antibody compared to Fc-treatedcontrols is presented in Table 34. The percent reduction in vasculararea reflects the anti-angiogenic potency of the antibody. (N/D=notdetermined)

TABLE 34 % Reduction in Vascular Area Relative to Fc Control AntibodyWild-Type Mice hAng-2 Mice H1H685P 39.7 N/D H1H690P 30.7 41.5 H1H691P30.4 N/D H1H696P 31.1 N/D H1H724N 32.2 33.2 H1H744N 35.8 50.5 Control I(Ab) 26.9 35.6

As shown in this Example, the selected anti-Ang-2 antibodies of thepresent invention substantially inhibited ocular angiogenesis in vivo,thus reflecting the likely anti-angiogenic potential of these antibodiesin other therapeutic contexts.

Example 13 Amino Acids of Ang-2 Important for Antibody Binding

To further characterize binding between hAng2 and anti-hAng2 mAbs of theinvention, seven variant hAng2-FD-mFc proteins were generated, eachcontaining a single point mutation. Amino acids selected for mutationwere based on the difference in sequence between hAng-2 and hAng-1 inthe region that interacts with hTie-2 (FIG. 1). In particular, aminoacids within the fibrinogen-like domain (FD) of Ang-2 which are believedto interact with Tie-2 based on crystal structure analysis, but whichdiffer from the corresponding amino acid in Ang-1, were individuallymutated to the corresponding hAng-1 residue. The results of this exampleindicate the amino acid residues of hAng-2 with which the Ang-2preferential binding antibodies interact. That is, if a particularresidue (or residues) of hAng-2 is/are changed to the correspondingresidue of hAng-1, and the binding of an Ang-2 preferential bindingantibody is substantially reduced, then it can be concluded that theantibody interacts with that particular residue(s) of hAng-2.

In this experiment, each of the seven hAng-2FD-mFc mutant proteins werecaptured (−147-283 RU) on an anti-mouse-Fc surface created throughdirect chemical coupling to a BIACORE™ chip. Then each Ang-2 antibody(or peptibody, as the case may be) at 100 nM was injected over thecaptured mFc-tagged hAng-2FD protein surface at a flowrate of 50 μl/minfor 180 sec, and the dissociation of variant hAng2-FD-mFc and antibodywas monitored in real time for 20 min at 25° C. Results are summarizedin Tables 35a-35d and FIG. 3.

TABLE 35a H1H685P H1H744N Mutated hAng-2 T ½ T ½ Amino Acid(s)^([1]) RUK_(D) (M) (min) RU K_(D) (M) (min) WT^([2]) 210.70 2.23E−11 1988 2133.98E−11 904 S-417-I 127.65 3.05E−11 1809 127 5.12E−11 1590 K-432-N152.68 1.40E−11 4468 137 4.87E−11 1690 I-434-M 235.95 1.79E−11 3600 2133.18E−11 2589 N-467-G 152.25 9.38E−12 6762 139 7.72E−11 1011 F-469-L101.16 1.38E−08 4 180 1.95E−10 237 Y-475-H 181.53 1.96E−10 289 2473.06E−10 136 S-480-P 161.13 2.05E−10 289 228 2.25E−11 2129

TABLE 35b Control I (Ab) Control II (Pb) Mutated hAng-2 T ½ T ½ AminoAcid(s)^([1]) RU K_(D) (M) (min) RU K_(D) (M) (min) WT^([2]) 195.254.69E−10 54.33 67.44 4.29E−10 39.86 S-417-I 142.96 5.79E−10 32.81 49.991.88E−10 36.38 K-432-N 189.69 3.49E−10 51.75 63.21 1.39E−10 42.34I-434-M 282.10 4.64E−10 48.80 89.15 1.36E−10 57.09 N-467-G 180.904.61E−10 44.66 60.94 1.54E−10 46.97 F-469-L 173.01 1.05E−09 25.13 46.732.40E−10 36.20 Y-475-H 170.05 1.15E−08 1.85 74.79 1.40E−10 54.12 S-480-P181.32 2.98E−09 13.36 71.90 1.79E−10 45.45

TABLE 35c Mutated hAng-2 Control III (Pb) Control V (Ab) AminoAcid(s)^([1]) RU K_(D) (M) T ½ (min) RU K_(D) (M) T ½ (min) WT^([2])80.33 2.07E−11 170.03 214.48 7.97E−10 48.43 S-417-I 57.13 5.31E−11114.81 126.45 2.40E−09 29.17 K-432-N 79.22 2.88E−11 200.94 149.148.48E−10 75.59 I-434-M 116.22 2.15E−10 62.77 214.75 2.23E−09 31.76N-467-G 74.64 8.90E−11 109.07 146.77 1.11E−09 55.66 F-469-L 72.662.74E−10 66.11 131.96 1.37E−08 1.46 Y-475-H 76.21 6.87E−09 4.11 260.932.66E−10 93.22 S-480-P 77.93 2.78E−09 11.69 177.10 3.47E−09 10.33

TABLE 35d Negative Control Mutated hAng-2 (irrelevant antibody) AminoAcid(s)^([1]) RU K_(D) (M) T½ (min) WT^([2]) 0.81 N/B N/B S-417-I −1.21N/B N/B K-432-N −0.38 N/B N/B I-434-M −1.31 N/B N/B N-467-G −1.09 N/BN/B F-469-L 0.32 N/B N/B Y-475-H −0.20 N/B N/B S-480-P −0.52 N/B N/B^([1])Amino acid numbering is based on the amino acid numbering of SEQID NO: 518. ^([2])WT = wild-type Ang-2FD-mFc construct. N/B = no bindingobserved.

For purposes of the present invention, an anti-Ang-2 antibody is deemedto interact with a particular Ang-2 amino acid residue if, when theresidue is mutated to the corresponding residue of Ang-1, the T1/2 ofdissociation is at least 5-fold less than the T1/2 of dissociationobserved for the wild-type construct under the experimental conditionsused in this Example. In view of this definition, antibody H1H685Pappears to be unique among the antibodies tested in that it interactswith F469, Y475 and S480. Since H1H685P is also unique because of itsstrong preferential binding to Ang-2 over Ang-1, it can be concludedthat F469, Y475 and S480 comprise an epitope that enables theimmunological distinction of Ang-2 from Ang-1. The otherantibodies/peptibodies tested in this experiment appear to interact withat most one or two of these residues; i.e., H1H744N and Control Iinteract with Y475; Control III interacts with Y475 and S480; andControl V interacts with F469. Interestingly, Control II, which wasshown to block both Ang-1 and Ang-2 binding to Tie-2 with equal potency,does not interact with any of the Ang-2-specific amino acids identifiedin this experiment.

The present invention is not to be limited in scope by the specificembodiments describe herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

What is claimed is:
 1. An isolated antibody or antigen-binding fragmentthereof that specifically binds human angiopoietin-2 (hAng-2), whereinthe antibody or antigen-binding fragment thereof comprises a heavy chainCDR-1 (HCDR1) having the amino acid sequence of SEQ ID NO:4, an HCDR-2having the amino acid sequence of SEQ ID NO:6, an HCDR-3 having theamino acid sequence of SEQ ID NO:8, a light chain CDR-1 (LCDR-1) havingthe amino acid sequence of SEQ ID NO:12, an LCDR-2 having the amino acidsequence of SEQ ID NO:14, and an LCDR-3 having the amino acid sequenceof SEQ ID NO:16.
 2. An isolated antibody or antigen-binding fragmentthereof that specifically binds human angiopoietin-2 (hAng-2), whereinthe antibody or antigen-binding fragment thereof comprises a heavy chainvariable region having the amino acid sequence of SEQ is NO: 18 and alight chain variable region having the amino acid sequence of SEQ NO:20.3. A pharmaceutical composition comprising the antibody orantigen-binding fragment of claim 1 and a pharmaceutically acceptablecarrier or diluent.
 4. The pharmaceutical composition of claim 3,further comprising a vascular endothelial cell growth factor (VEGF)antagonist.
 5. The pharmaceutical composition of claim 4, wherein theVEGF antagonist is selected from the group consisting of an anti-VEGFantibody, a small molecule kinase inhibitor of a VEGF receptor and aVEGF-inhibiting fusion protein.
 6. A pharmaceutical compositioncomprising the antibody or antigen-binding fragment of claim 2 and apharmaceutically acceptable carrier or diluent.
 7. The pharmaceuticalcomposition of claim 6, further comprising a vascular endothelial cellgrowth factor (VEGF) antagonist.
 8. The pharmaceutical composition ofclaim 7, wherein the VEGF antagonist is selected from the groupconsisting of an anti-VEGF antibody, a small molecule kinase inhibitorof VEGF receptor and a VEGF-inhibiting fusion protein.
 9. An isolatedhuman antibody or antigen-binding fragment thereof comprising at leastone antibody variable domain, wherein the antibody or antigen-bindingfragment binds to the same epitope on hAng-2 as an antibody whichcomprises the complementarity determining regions (CDRs) of a heavychain variable region (HCVR) having the amino acid sequence of SEQ IDN0:18, and the CDRs of a light chain variable region (LCVR) having theamino acid sequence of SEQ ID NO:20.
 10. An isolated human antibody orantigen-binding fragment thereof comprising at least one antibodyvariable domain, wherein the antibody or antigen-binding fragment bindsto the same epitope on hAng-2 as an antibody which comprises a heavychain CDR-1 (HCDR1) having the amino acid sequence of SEQ ID NO:4, anHCDR-2 having the amino acid sequence of SEQ ID NO:6, an HCDR-3 havingthe amino acid sequence of SEQ ID NO:8, a light chain CDR-1 (LCDR-1)having the amino acid sequence of SEQ ID NO:12, an LCDR-2 having theamino acid sequence of SEQ ID NO:14, and an LCDR-3 having the amino acidsequence of SEQ ID NO:16.